Mutant interleukin-2 polypeptides

ABSTRACT

The present invention generally relates to mutant interleukin-2 polypeptides that exhibit reduced affinity to the α-subunit of the IL-2 receptor, for use as immunotherapeutic agents. In addition, the invention relates to immunoconjugates comprising said mutant IL-2 polypeptides, polynucleotide molecules encoding the mutant IL-2 polypeptides or immunoconjugates, and vectors and host cells comprising such polynucleotide molecules. The invention further relates to methods for producing the mutant IL-2 polypeptides or immunoconjugates, pharmaceutical compositions comprising the same, and uses thereof.

FIELD OF THE INVENTION

The present invention generally relates to mutant interleukin-2polypeptides. More particularly, the inventions concerns mutant IL-2polypeptides that exhibit improved properties for use asimmunotherapeutic agents. In addition, the invention relates toimmunoconjugates comprising said mutant IL-2 polypeptides,polynucleotide molecules encoding the mutant IL-2 polypeptides orimmunoconjugates, and vectors and host cells comprising suchpolynucleotide molecules. The invention further relates to methods forproducing the mutant IL-2 polypeptides or immunoconjugates,pharmaceutical compositions comprising the same, and uses thereof.

BACKGROUND

Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a15.5 kDa globular glycoprotein playing a central role in lymphocytegeneration, survival and homeostasis. It has a length of 133 amino acidsand consists of four antiparallel, amphiphatic α-helices that form aquaternary structure indispensable of its function (Smith, Science 240,1169-76 (1988); Bazan, Science 257, 410-413 (1992)). Sequences of IL-2from different species are found under NCBI Ref Seq Nos. NP000577(human), NP032392 (mouse), NP446288 (rat) or NP517425 (chimpanzee).

IL-2 mediates its action by binding to IL-2 receptors (IL-2R), whichconsist of up to three individual subunits, the different association ofwhich can produce receptor forms that differ in their affinity to IL-2.Association of the an (CD25), β (CD122), and γ (γ_(c), CD132) subunitsresults in a trimeric, high-affinity receptor for IL-2. Dimeric IL-2receptor consisting of the β and γ subunits is termedintermediate-affinity IL-2R. The a subunit forms the monomeric lowaffinity IL-2 receptor. Although the dimeric intermediate-affinity IL-2receptor binds IL-2 with approximately 100-fold lower affinity than thetrimeric high-affinity receptor, both the dimeric and the trimeric IL-2receptor variants are able to transmit signal upon IL-2 binding (Minamiet al., Annu Rev Immunol 11, 245-268 (1993)). Hence, the α-subunit,CD25, is not essential for IL-2 signalling. It confers high-affinitybinding to its receptor, whereas the β subunit, CD122, and the γ-subunitare crucial for signal transduction (Krieg et al., Proc Natl Acad Sci107, 11906-11 (2010)). Trimeric IL-2 receptors including CD25 areexpressed by (resting) CD4⁺ forkhead box P3 (FoxP3)⁺ regulatory T(T_(reg)) cells. They are also transiently induced on conventionalactivated T cells, whereas in the resting state these cells express onlydimeric IL-2 receptors. T_(reg) cells consistently express the highestlevel of CD25 in vivo (Fontenot et al., Nature Immunol 6, 1142-51(2005)).

IL-2 is synthesized mainly by activated T-cells, in particular CD⁺helper T cells. It stimulates the proliferation and differentiation of Tcells, induces the generation of cytotoxic T lymphocytes (CTLs) and thedifferentiation of peripheral blood lymphocytes to cytotoxic cells andlymphokine-activated killer (LAK) cells, promotes cytokine and cytolyticmolecule expression by T cells, facilitates the proliferation anddifferentiation of B-cells and the synthesis of immunoglobulin byB-cells, and stimulates the generation, proliferation and activation ofnatural killer (NK) cells (reviewed e.g. in Waldmann, Nat Rev Immunol 6,595-601 (2009); Olejniczak and Kasprzak, Med Sci Monit 14, RA179-89(2008); Malek, Annu Rev Immunol 26, 453-79 (2008)).

Its ability to expand lymphocyte populations in vivo and to increase theeffector functions of these cells confers antitumor effects to IL-2,making IL-2 immunotherapy an attractive treatment option for certainmetastatic cancers. Consequently, high-dose IL-2 treatment has beenapproved for use in patients with metastatic renal-cell carcinoma andmalignant melanoma.

However, IL-2 has a dual function in the immune response in that it notonly mediates expansion and activity of effector cells, but also iscrucially involved in maintaining peripheral immune tolerance.

A major mechanism underlying peripheral self-tolerance is IL-2 inducedactivation-induced cell death (AICD) in T cells. AICD is a process bywhich fully activated T cells undergo programmed cell death throughengagement of cell surface-expressed death receptors such as CD95 (alsoknown as Fas) or the TNF receptor. When antigen-activated T cellsexpressing a high-affinity IL-2 receptor (after previous exposure toIL-2) during proliferation are re-stimulated with antigen via the T cellreceptor (TCR)/CD3 complex, the expression of Fas ligand (FasL) and/ortumor necrosis factor (TNF) is induced, making the cells susceptible forFas-mediated apoptosis. This process is IL-2 dependent (Lenardo, Nature353, 858-61 (1991)) and mediated via STAT5. By the process of AICD in Tlymphocytes tolerance can not only be established to self-antigens, butalso to persistent antigens that are clearly not part of the host'smakeup, such as tumor antigens.

Moreover, IL-2 is also involved in the maintenance of peripheral CD4⁺CD25⁺ regulatory T (T_(reg)) cells (Fontenot et al., Nature Immunol 6,1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005);Maloy and Powrie, Nature Immunol 6, 1171-72 (2005), which are also knownas suppressor T cells. They suppress effector T cells from destroyingtheir (self-)target, either through cell-cell contact by inhibiting Tcell help and activation, or through release of immunosuppressivecytokines such as IL-10 or TGF-β. Depletion of T_(reg) cells was shownto enhance IL-2 induced anti-tumor immunity (Imai et al., Cancer Sci 98,416-23 (2007)).

Therefore, IL-2 is not optimal for inhibiting tumor growth, because inthe presence of IL-2 either the CTLs generated might recognize the tumoras self and undergo AICD or the immune response might be inhibited byIL-2 dependent T_(reg) cells.

A further concern in relation to IL-2 immunotherapy are the side effectsproduced by recombinant human IL-2 treatment. Patients receivinghigh-dose IL-2 treatment frequently experience severe cardiovascular,pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous,haematological and systemic adverse events, which require intensivemonitoring and in-patient management. The majority of these side effectscan be explained by the development of so-called vascular (or capillary)leak syndrome (VLS), a pathological increase in vascular permeabilityleading to fluid extravasation in multiple organs (causing e.g.pulmonary and cutaneous edema and liver cell damage) and intravascularfluid depletion (causing a drop in blood pressure and compensatoryincrease in heart rate). There is no treatment of VLS other thanwithdrawal of IL-2. Low-dose IL-2 regimens have been tested in patientsto avoid VLS, however, at the expense of suboptimal therapeutic results.VLS was believed to be caused by the release of proinflammatorycytokines, such as tumor necrosis factor (TNF)-α from IL-2-activated NKcells, however it has recently been shown that IL-2-induced pulmonaryedema resulted from direct binding of IL-2 to lung endothelial cells,which expressed low to intermediate levels of functional αβγ IL-2receptors (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

Several approaches have been taken to overcome these problems associatedwith IL-2 immunotherapy. For example, it has been found that thecombination of IL-2 with certain anti-IL-2 monoclonal antibodiesenhances treatment effects of IL-2 in vivo (Kamimura et al., J Immunol177, 306-14 (2006); Boyman et al., Science 311, 1924-27 (2006)). In analternative approach, IL-2 has been mutated in various ways to reduceits toxicity and/or increase its efficacy. Hu et al. (Blood 101,4853-4861 (2003), US Pat. Publ. No. 2003/0124678) have substituted thearginine residue in position 38 of IL-2 by tryptophan to eliminateIL-2's vasopenneability activity. Shanafelt et al. (Nature Biotechnol18, 1197-1202 (2000)) have mutated asparagine 88 to arginine to enhanceselectivity for T cells over NK cells. Heaton et al. (Cancer Res 53,2597-602 (1993); U.S. Pat. No. 5,229,109) have introduced two mutations,Arg38Ala and Phe42Lys, to reduce the secretion of proinflammatorycytokines from NK cells. Gillies et al. (US Pat. Publ. No. 2007/0036752)have substituted three residues of IL-2 (Asp20Thr, Asn88Arg, andGln126Asp) that contribute to affinity for the intermediate-affinityIL-2 receptor to reduce VLS. Gillies t al. (WO 2008/0034473) have alsomutated the interface of IL-2 with CD25 by amino acid substitutionArg38Trp and Phe42Lys to reduce interaction with CD25 and activation ofT_(reg) cells for enhancing efficacy. To the same aim, Wittrup et al.(WO 2009/061853) have produced IL-2 mutants that have enhanced affinityto CD25, but do not activate the receptor, thus act as antagonists. Themutations introduced were aimed at disrupting the interaction with theβ- and/or γ-subunit of the receptor.

However, none of the known IL-2 mutants was shown to overcome all of theabove-mentioned problems associated with IL-2 immunotherapy, namelytoxicity caused by the induction of VLS, tumor tolerance caused by theinduction of AICD, and immunosuppression caused by activation of T_(reg)cells. Thus there remains a need in the art to further enhance thetherapeutic usefulness of IL-2 proteins.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the recognition that theinteraction of IL-2 with the α-subunit of the trimeric, high-affinityIL-2 receptor is responsible for the problems associated with IL-2immunotherapy.

Accordingly, in a first aspect the invention provides a mutantinterleukin-2 (IL-2) polypeptide comprising a first amino acid mutationthat abolishes or reduces affinity of the mutant IL-2 polypeptide to thehigh-affinity IL-2 receptor and preserves affinity of the mutant IL-2polypeptide to the intermediate-affinity IL-2 receptor, each compared toa wild-type IL-2 polypeptide. In one embodiment said first amino acidmutation is at a position corresponding to residue 72 of human IL-2 (SEQID NO:1). In one embodiment said first amino acid mutation is an aminoacid substitution, selected from the group of L72G, L72A, L72S, L72T,L72Q, L72E, L72N, L72D, L72R, and L72K. In a more specific embodimentsaid first amino acid mutation is the amino acid substitution L72G. Incertain embodiments the mutant IL-2 polypeptide comprises a second aminoacid mutation that abolishes or reduces affinity of the mutant IL-2polypeptide to the high-affinity IL-2 receptor and preserves affinity ofthe mutant IL-2 polypeptide to the intermediate-affinity IL-2 receptor,each compared to a wild-type IL-2 polypeptide. In one embodiment saidsecond amino acid mutation is at a position selected from the positionscorresponding to residue 35, 38, 42, 43, and 45 of human IL-2 (SEQ IDNO:1). In a specific embodiment said second amino acid mutation is at aposition corresponding to residue 42 of human IL-2 (SEQ ID NO:1). In amore specific embodiment said second amino acid mutation is an aminoacid substitution, selected from the group of F42A, F42G, F42S, F42T,F42Q, F42E, F42N, F42D, F42R, and F42K. In an even more specificembodiment said second amino acid mutation is the amino acidsubstitution F42A. In certain embodiments the mutant interleukin-2polypeptide comprises a third amino acid mutation that abolishes orreduces affinity of the mutant IL-2 polypeptide to the high-affinityIL-2 receptor and preserves affinity of the mutant IL-2 polypeptide tothe intermediate-affinity IL-2 receptor, each compared to a wild-typeIL-2 polypeptide. In a particular embodiment, the mutant interleukin-2polypeptide comprises three amino acid mutations that abolish or reduceaffinity of the mutant IL-2 polypeptide to the high-affinity IL-2receptor and preserve affinity of the mutant IL-2 polypeptide to theintermediate-affinity IL-2 receptor, each compared to a wild-type IL-2polypeptide, wherein said three amino acid mutations are at positionscorresponding to residue 42, 45, and 72 of human IL-2 (SEQ ID NO:1). Inone embodiment said three amino acid mutations are amino acidsubstitutions selected from the group of F42A, F42G, F42S, F42T, F42Q,F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N,Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R,and L72K. In a specific embodiment said three amino acid mutations arethe amino acid substitutions F42A, Y45A and L72G. In certain embodimentsthe mutant interleukin-2 polypeptide further comprises an amino acidmutation which eliminates the O-glycosylation site of IL-2 at a positioncorresponding to residue 3 of human IL-2 (SEQ ID NO:1). In oneembodiment said amino acid mutation which eliminates the O-glycosylationsite of IL-2 at a position corresponding to residue 3 of human IL-2 isan amino acid substitution selected from the group of T3A, T3G, T3Q,T3E, T3N, T3D, T3R, T3K, and T3P. In a specific embodiment the aminoacid mutation which eliminates the O-glycosylation site of IL-2 at aposition corresponding to residue 3 of human IL-2 is T3A. In certainembodiments the mutant IL-2 polypeptide is essentially a full-lengthIL-2 molecule, particularly a human full-length IL-2 molecule.

The invention further provides for a mutant interleukin-2 polypeptidelinked to a non-IL-2 moiety. In certain embodiments said non-IL-2 moietyis a targeting moiety. In certain embodiments said non-IL-2 moiety is anantigen binding moiety. In one embodiment said antigen binding moiety isan antibody. In another embodiment said antigen binding moiety is anantibody fragment. In a more specific embodiment said antigen bindingmoiety is selected from a Fab molecule and a scFv molecule. In aparticular embodiment said antigen binding moiety is a Fab molecule. Inanother embodiment said antigen binding moiety is a scFv molecule. Inparticular embodiments the mutant IL-2 polypeptide is linked to a firstand a second non-IL-2 moiety. In one such embodiment the mutantinterleukin-2 polypeptide shares a carboxy-terminal peptide bond withsaid first non-IL-2 moiety and an amino-terminal peptide bond with saidsecond non-IL-2 moiety. In one embodiment said antigen binding moiety isan immunoglobulin molecule. In a more specific embodiment said antigenbinding moiety is an IgG class, particularly an IgG₁ subclass,immunoglobulin molecule. In certain embodiments said antigen bindingmoiety is directed to an antigen presented on a tumor cell or in a tumorcell environment, particularly an antigen selected from the group ofFibroblast Activation Protein (FAP), the A1 domain of Tenascin-C (TNCA1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B ofFibionectin (EDB), Carcinoembryonic Antigen (CEA) and theMelanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).

Also provided by the invention is an immunoconjugate comprising a mutantIL-2 polypeptide as described herein, and an antigen binding moiety. Inone embodiment of the immunoconjugate according to the invention themutant IL-2 polypeptide shares an amino- or carboxy-terminalpeptide-bond With said antigen binding moiety. In particular embodimentsthe immunoconjugate comprises as first and a second antigen bindingmoiety. In one such embodiment the mutant IL-2 polypeptide comprised inthe immunoconjugate according to the invention shares an amino- orcarboxy-terminal peptide bond with a first antigen binding moiety and asecond antigen binding moiety shares an amino- or carboxy-terminalpeptide bond with either i) the mutant IL-2 polypeptide or ii) saidfirst antigen binding moiety. In one embodiment the antigen bindingmoiety comprised in the immunoconjugate according to the invention is anantibody, in another embodiment said antigen binding moiety is anantibody fragment. In a specific embodiment said antigen binding moietyis selected from a Fab molecule and a scFv molecule. In a particularembodiment said antigen binding moiety is a Fab molecule. In anotherparticular embodiment said antigen binding moiety is an immunoglobulinmolecule. In a more specific embodiment said antigen binding moiety isan IgG class, particularly an IgG, subclass, immunoglobulin molecule. Incertain embodiments said antigen binding moiety is directed to anantigen presented on a tumor cell or in a tumor cell environment,particularly an antigen selected from the group of Fibroblast ActivationProtein (FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain ofTenascin-C (TNC A2), the Extra Domain B of Fibronectin (EDB),Carcinoembryonic Antigen (CEA) and the Melanoma-associated ChondroitinSulfate Proteoglycan (MCSP).

The invention further provides isolated polynucleotides encoding amutant IL-2 polypeptide or an immunoconjugate as described herein,expression vectors comprising said polynucleotides, and host cellscomprising the polynucleotides or the expression vectors.

Also provided is a method of producing a mutant IL-2 polypeptide or animmunoconjugate as described herein, comprising culturing the host celldescribed herein under conditions suitable for the expression of themutant IL-2 polypeptide or immunoconjugate as described herein andisolating the mutant IL-2 polypeptide or immunoconjugate as describedherein; a mutant IL-2 polypeptide or an immunoconjugate produced by themethod described herein, a pharmaceutical composition comprising amutant IL-2 polypeptide or an immunoconjugate as described herein and apharmaceutically acceptable carrier, and methods of using a mutant IL-2polypeptide or an immunoconjugate as described herein.

In particular, the invention encompasses a mutant IL-2 polypeptide or animmunoconjugate as described herein for use in the treatment of adisease in an individual in need thereof. In a particular embodimentsaid disease is cancer. In a particular embodiment the individual is ahuman.

Also encompassed by the invention is the use of the mutant IL-2polypeptide or immunoconjugate as described herein for the manufactureof a medicament for treating a disease in an individual in need thereof.

Further provided is a method of treating disease in an individual,comprising administering to said individual a therapeutically effectiveamount of a composition comprising a mutant IL-2 polypeptide or animmunoconjugate as described herein. Said disease preferably is cancer.

Also provided is a method of stimulating the immune system of anindividual, comprising administering to said individual an effectiveamount of a composition comprising the mutant IL-2 polypeptide orimmunoconjugate described herein in a pharmaceutically acceptable form.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

The term “interleukin-2” or “IL-2” as used herein, refers to any nativeIL-2 from any vertebrate source, including mammals such as primates(e.g. humans) and rodents (e.g., mice and rats), unless otherwiseindicated. The term encompasses unprocessed IL-2 as well as any form ofIL-2 that results from processing in the cell. The term also encompassesnaturally occurring variants of IL-2, e.g. splice variants or allelicvariants. The amino acid sequence of an exemplary human IL-2 is shown inSEQ ID NO: 1. Unprocessed human IL-2 additionally comprises anN-terminal 20 amino acid signal peptide having the sequence of SEQ IDNO: 272, which is absent in the mature IL-2 molecule.

The term “IL-2 mutant” or “mutant IL-2 polypeptide” as used herein isintended to encompass any mutant forms of various forms of the IL-2molecule including full-length IL-2, truncated forms of IL-2 and formswhere IL-2 is linked to another molecule such as by fusion or chemicalconjugation. “Full-length” when used in reference to IL-2 is intended tomean the mature, natural length IL-2 molecule. For example, full-lengthhuman IL-2 refers to a molecule that has 133 amino acids (see e.g. SEQID NO: 1). The various forms of IL-2 mutants are characterized in havinga at least one amino acid mutation affecting the interaction of IL-2with CD25. This mutation may involve substitution, deletion, truncationor modification of the wild-type amino acid residue normally located atthat position. Mutants obtained by amino acid substitution arepreferred. Unless otherwise indicated, an IL-2 mutant may be referred toherein as an IL-2 mutant peptide sequence, an IL-2 mutant polypeptide,IL-2 mutant protein or IL-2 mutant analog.

Designation of various forms of IL-2 is herein made with respect to thesequence shown in SEQ ID NO: 1. Various designations may be used hereinto indicate the same mutation. For example a mutation from phenylalanineat position 42 to alanine can be indicated as 42A, A42, A₄₂, F42A, orPhe42Ala.

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto CD25. Amino acid sequence deletions and insertions include amino-and/or carboxy-terminal deletions and insertions of amino acids. Anexample of a terminal deletion is the deletion of the alanine residue inposition 1 of full-length human IL-2. Preferred amino acid mutations areamino acid substitutions. For the purpose of altering e.g. the bindingcharacteristics of an IL-2 polypeptide, non-conservative amino acidsubstitutions, i.e. replacing one amino acid with another amino acidhaving different structural and/or chemical properties, are particularlypreferred. Preferred amino acid substitions include replacing ahydrophobic by a hydrophilic amino acid. Amino acid substitutionsinclude replacement by non-naturally occurring amino acids or bynaturally occurring amino acid derivatives of the twenty standard aminoacids (e.g. 4-hydroxyproline, 3-methylhistidine, omithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful.

As used herein, a “wild-type” form of IL-2 is a form of IL-2 that isotherwise the same as the mutant IL-2 polypeptide except that thewild-type form has a wild-type amino acid at each amino acid position ofthe mutant IL-2 polypeptide. For example, if the IL-2 mutant is thefull-length IL-2 (i.e. IL-2 not fused or conjugated to any othermolecule), the wild-type form of this mutant is full-length native IL-2.If the IL-2 mutant is a fusion between IL-2 and another polypeptideencoded downstream of IL-2 (e.g. an antibody chain) the wild-type formof this IL-2 mutant is IL-2 with a wild-type amino acid sequence fusedto the same downstream polypeptide. Furthermore, if the IL-2 mutant is atruncated form of IL-2 (the mutated or modified sequence within thenon-truncated portion of IL-2) then the wild-type form of this IL-2mutant is a similarly truncated IL-2 that has a wild-type sequence. Forthe purpose of comparing IL-2 receptor binding affinity or biologicalactivity of various forms of IL-2 mutants to the corresponding wild-typeform of IL-2, the term wild-type encompasses forms of IL-2 comprisingone or more amino acid mutation that does not affect IL-2 receptorbinding compared to the naturally occurring, native IL-2, such as e.g. asubstitution of cysteine at a position corresponding to residue 125 ofhuman IL-2 to alanine. In some embodiments wild-type IL-2 for thepurpose of the present invention comprises the amino acid substitutionC125A (see SEQ ID NO: 3). In certain embodiments according to theinvention the wild-type IL-2 polypeptide to which the mutant IL-2polypeptide is compared comprises the amino acid sequence of SEQ IDNO: 1. In other embodiments the wild-type IL-2 polypeptide to which themutant IL-2 polypeptide is compared comprises the amino acid sequence ofSEQ ID NO: 3.

The term “CD25” or “α-subunit of the IL-2 receptor” as used herein,refers to any native CD25 from any vertebrate source, including mammalssuch as primates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length”, unprocessedCD25 as well as any form of CD25 that results from processing in thecell. The term also encompasses naturally occurring variants of CD25,e.g. splice variants or allelic variants. In certain embodiments CD25 ishuman CD25. The amino acid sequence of an exemplary human CD25 (withsignal sequence, Avi-tag and His-tag) is shown in SEQ ID NO: 278.

The term “high-affinity IL-2 receptor” as used herein refers to theheterotrimeric form of the IL-2 receptor, consisting of the receptorγ-subunit (also known as common cytokine receptor γ-subunit, γ_(c), orCD132), the receptor α-subunit (also known as CD122 or p70) and thereceptor α-subunit (also known as CD25 or p55). The term“intermediate-affinity IL-2 receptor” by contrast refers to the IL-2receptor including only the γ-subunit and the α-subunit, without theα-subunit (for a review see e.g. Olejniczak and Kasprzak, Med Sci Monit14, RA179-189 (2008)).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., areceptor) and its binding partner (e.g., a ligand). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., receptor and a ligand). The affinity of a molecule-Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)), which is the ratio of dissociation and associationrate constants (k_(off) and k_(on), respectively). Thus, equivalentaffinities may comprise different rate constants, as long as the ratioof the rate constants remains the same. Affinity can be measured by wellestablished methods known in the art, including those described herein.

The affinity of the mutant or wild-type IL-2 polypeptide for variousforms of the IL-2 receptor can be determined in accordance with themethod set forth in the Examples by surface plasmon resonance (SPR),using standard instrumentation such as a BIAcore instrument (GEHealthcare) and receptor subunits such as may be obtained by recombinantexpression (see e.g. Shanafelt et al., Nature Biotechnol 18, 1197-1202(2000)). Alternatively, binding affinity of IL-2 mutants for differentforms of the IL-2 receptor may be evaluated using cell lines known toexpress one or the other such form of the receptor. Specificillustrative and exemplary embodiments for measuring binding affinityare described hereinafter.

By “regulatory. T cell” or “T_(reg) cell” is meant a specialized type ofCD4⁺ T cell that can suppress the responses of other T cells. T_(reg)cells are characterized by expression of the α-subunit of the IL-2receptor (CD25) and the transcription factor forkhead box P3 (FOXP3)(Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play a critical rolein the induction and maintenance of peripheral self-tolerance toantigens, including those expressed by tumors. T_(reg) cells requireIL-2 for their function and development and induction of theirsuppressive characteristics.

As used herein, the term “effector cells” refers to a population oflymphocytes that mediate the cytotoxic effects of IL-2. Effector cellsinclude effector T cells such as CD8⁺ cytotoxic T cells, NK cells,lymphokine-activated killer (LAK) cells and macrophages/monocytes.

As used herein, the term “antigen binding moiety” refers to apolypeptide molecule that specifically binds to an antigenicdeterminant. In one embodiment, an antigen binding moiety is able todirect the entity to which it is attached (e.g. a cytokine or a secondantigen binding moiety) to a target site, for example to a specific typeof tumor cell or tumor stroma bearing the antigenic determinant. Antigenbinding moieties include antibodies and fragments thereof as furtherdefined herein. Preferred antigen binding moieties include an antigenbinding domain of an antibody, comprising an antibody heavy chainvariable region and an antibody light chain variable region. In certainembodiments, the antigen binding moieties may include antibody constantregions as further defined herein and known in the art. Useful heavychain constant regions include any of the five isotypes: α, δ, ε, γ, orβ. Useful light chain constant regions include-any of the two isotypes:κ and λ.

By “specifically binds” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antigen binding moiety to bind to aspecific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)).

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antigen binding moiety binds, forming an antigen bindingmoiety-antigen complex. Useful antigenic determinants can be found, forexample, on the surfaces of tumor cells, on the surfaces ofvirus-infected cells, on the surfaces of other diseased cells, free inblood serum, and/or in the extracellular matrix (ECM).

As used herein, term “polypeptide” refers to a molecule composed ofmonomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain of two ormore amino acids, and does not refer to a specific length of theproduct. Thus, peptides, dipeptides, tripeptides, oligopeptides,“protein,” “amino acid chain,” or any other term used to refer to achain of two or more amino acids, are included within the definition of“polypeptide,” and the term “polypeptide” may be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide may be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Itmay be generated in any manner, including by chemical synthesis. Apolypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded.

By an “isolated” polypeptide or a variant, or derivative thereof isintended a polypeptide that is not in its natural milieu. No particularlevel of purification is required. For example, an isolated polypeptidecan be removed from its native or natural environment. Recombinantlyproduced polypeptides and proteins expressed in host cells areconsidered isolated for the purpose of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “polynucleotide” refers to an isolated nucleic acid molecule orconstruct, e.g. messenger RNA (mRNA), virally-derived RNA, or plasmidDNA (pDNA). A polynucleotide may comprise a conventional phosphodiesterbond or a non-conventional bond (e.g. an amide bond, such as found inpeptide nucleic acids (PNA). The term “nucleic acid molecule” refers toany one or more nucleic acid segments, e.g. DNA or RNA fragments,present in a polynucleotide.

By “isolated” nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding atherapeutic polypeptide contained in a vector is considered isolated forthe purposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. An isolated polynucleotide includes apolynucleotide molecule contained in cells that ordinarily contain thepolynucleotide molecule, but the polynucleotide molecule is presentextrachromosomally or at a chromosomal location that is different fromits natural chromosomal location. Isolated RNA molecules include in vivoor in vitro RNA transcripts of the present invention, as well aspositive and negative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition, apolynucleotide or a nucleic acid may be or may include a regulatoryelement such as a promoter, ribosome binding site, or a transcriptionterminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at the5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence. As a practical matter,whether any particular polynucleotide sequence is at least 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of thepresent invention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.ALIGN-2).

The term “expression cassette” refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in atarget cell. The recombinant expression cassette can be incorporatedinto a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, ornucleic acid fragment. Typically, the recombinant expression cassetteportion of an expression vector includes, among other sequences, anucleic acid sequence to be transcribed and a promoter. In certainembodiments, the expression cassette of the invention comprisespolynucleotide sequences that encode mutant IL-2 polypeptides orimmunoconjugates of the invention or fragments thereof.

The term “vector” or “expression vector” is synonymous with “expressionconstruct” and refers to a DNA molecule that is used to introduce anddirect the expression of a specific gene to which it is operablyassociated in a target cell. The term includes the vector as aself-replicating nucleic acid structure as well as the vectorincorporated into the genome of a host cell into which it has beenintroduced. The expression vector of the present invention comprises anexpression cassette. Expression vectors allow transcription of largeamounts of stable mRNA. Once the expression vector is inside the targetcell, the ribonucleic acid molecule or protein that is encoded by thegene is produced by the cellular transcription and/or translationmachinery. In one embodiment, the expression vector of the inventioncomprises an expression cassette that comprises polynucleotide sequencesthat encode mutant IL-2 polypeptides or immunoconjugates of theinvention or fragments thereof.

The term “artificial” refers to a synthetic, or non-host cell derivedcomposition, e.g. a chemically-synthesized oligonucleotide.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages.

Progeny may not be completely identical in nucleic acid content to aparent cell, but may contain mutations. Mutant progeny that have thesame function or biological activity as screened or selected for in theoriginally transformed cell are included herein.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen binding activity.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), and multispecific antibodies formed from antibodyfragments. For a review of certain antibody fragments, see Hudson etal., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g.Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994);see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. Fordiscussion of Fab and F(ab′)₂ fragments comprising salvage receptorbinding epitope residues and having increased in vivo half-life, seeU.S. Pat. No. 5,869,046. Diabodies are antibody fragments with twoantigen binding sites that may be bivalent or bispecific. See, forexample, EP 404,097; WO 1993/01161; Hudson et al., Nat Med 9, 129-134(2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448(1993). Triabodies and tetrabodies are also described in Hudson et al.,Nat Med 9, 129-134 (2003). Antibody fragments can be made byvarious-techniques, including but not limited to proteolytic digestionof an intact antibody as well as production by recombinant host cells(e.g. E. coli or phage), as described herein.

The term “immunoglobulin molecule” refers to a protein having thestructure of a naturally occurring antibody. For example,immunoglobulins of the IgG class are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavy chainsthat are disulfide-bonded. From N- to C-terminus, each heavy chain has avariable region (VH), also called a variable heavy domain or a heavychain variable domain, followed by three constant domains (CH1, CH2, andCH3), also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fiveclasses, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subclasses, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcdomain, linked via the immunoglobulin hinge region.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Preferably, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to confer antigenbinding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The appropriate amino acid residues which encompass the CDRs asdefined by each of the above cited references are set forth below inTable 1 as a comparison. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table 1 refers to theCDRs as defmed by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

The polypeptide sequences of the sequence listing (i.e., SEQ ID NOs: 23,25, 27, 29, 31, 33, etc.) are not numbered according to the Kabatnumbering system. However, it is well within the ordinary skill of onein the art to convert the numbering of the sequences of the SequenceListing to Kabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of an IgGheavy chain might vary slightly, the human IgG heavy chain Fc region isusually defined to extend from Cys226, or from Pro230, to thecarboxyl-terminus of the heavy chain. However, the C-terminal lysine(Lys447) of the Fc region may or may not be present.

A “modification promoting heterodimerization” is a manipulation of thepeptide backbone or the post-translational modifications of apolypeptide, e.g. an immunoglobulin heavy chain, that reduces orprevents the association of the polypeptide with an identicalpolypeptide to form a homodimer. A modification promotingheterodimerization as used herein particularly includes separatemodifications made to each of two polypeptides desired to form a dimer,wherein the modifications are complementary to each other so as topromote association of the two polypeptides. For example, a modificationpromoting heterodimerization may alter the structure or charge of one orboth of the polypeptides desired to form a dimer so as to make theirassociation sterically or electrostatically favorable, respectively.Heterodimerization occurs between two non-identical polypeptides, suchas two immunoglobulin heavy chains wherein further immunoconjugatecomponents fused to each of the heavy chains (e.g. IL-2 polypeptide) arenot the same. In the immunoconjugates of the present invention, themodification promoting heterodimerization is in the heavy chain(s),specifically in the Fc domain, of an immunoglobulin molecule. In someembodiments the modification promoting heterodimerization comprises anamino acid mutation, specifically an amino acid substitution. In aparticular embodiment, the modification promoting heterodimerizationcomprises a separate amino acid mutation, specifically an amino acidsubstitution, in each of the two immunoglobulin heavy chains.

The term “effector functions” when used in reference to antibodies referto those biological activities attributable to the Fc region of anantibody, which vary with the antibody isotype. Examples of antibodyeffector functions include: C1q binding and complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), cytokine secretion, down regulation of cell surfacereceptors (e.g. B cell receptor), and B cell activation.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89).

As used herein, the terms “engineer, engineered, engineering”, areconsidered to include my manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches.

As used herein, the term “immunoconjugate” refers to a polypeptidemolecule that includes at least one IL-2 moiety and at least one antigenbinding moiety. In certain embodiments, the immunoconjugate comprises atleast one IL-2 moiety, and at least two antigen binding moieties.Particular immunoconjugates according to the invention essentiallyconsist of one IL-2 moiety and two antigen binding moieties joined byone or more linker sequences. The antigen binding moiety can be joinedto the IL-2 moiety by a variety of interactions and in a variety ofconfigurations as described herein.

As used herein, the term “control antigen binding moiety” refers to anantigen binding moiety as it would exist free of other antigen bindingmoieties and effector moieties. For example, when comparing anFab-IL2-Fab immunoconjugate of the invention with a control antigenbinding moiety, the control antigen binding moiety is free Fab, whereinthe Fab-IL2-Fab immunoconjugate and the free Fab molecule can bothspecifically bind to the same antigen determinant.

As used herein, the terms “first” and “second” with respect to antigenbinding moieties etc., are used for convenience of distinguishing whenthere is more than one of each type of moiety. Use of these terms is notintended to confer a specific order or orientation of theimmunoconjugate unless explicitly so stated.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Preferably, the individual orsubject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe composition would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical composition, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect, pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention aims at providing a mutant IL-2 polypeptide havingimproved properties for immunotherapy. In particular the invention aimsat eliminating pharmacological properties of IL-2 that contribute totoxicity but are not essential for efficacy of IL-2. As discussed above,different forms of the IL-2 receptor consist of different subunits andexhibit different affinities for IL-2. The intermediate-affinity IL-2receptor, consisting of the β and γ receptor subunits, is expressed onresting effector cells and is sufficient for IL-2 signaling. Thehigh-affinity IL-2 receptor, additionally comprising the α-subunit ofthe receptor, is mainly expressed on regulatory T (T_(reg)) cells aswell as on activated effector cells where its engagement by IL-2 canpromote T_(reg) cell-mediated immunosuppression or activation-inducedcell death (AICD), respectively. Thus, without wishing to be bound bytheory, reducing or abolishing the affinity of IL-2 to the α-subunit ofthe IL-2 receptor should reduce IL-2 induced downregulation of effectorcell function by regulatory T cells and development of tumor toleranceby the process of AICD. On the other hand, maintaining the affinity tothe intermediate-affinity IL-2 receptor should preserve the induction ofproliferation and activation of effector cells like NK and T cells byIL-2.

Several IL-2 mutants already exist in the art, however, the inventorshave found novel amino acid mutations of the IL-2 polypeptide andcombinations thereof that are particularly suitable to confer to IL-2the desired characteristics for immunotherapy.

In a first aspect the invention provides a mutant interleukin-2 (IL-2)polypeptide comprising an amino acid mutation that abolishes or reducesaffinity of the mutant IL-2 polypeptide to the α-subunit of the IL-2receptor and preserves affinity of the mutant IL-2 polypeptide to theintermediate-affinity IL-2 receptor each compared to a wild-type IL-2polypeptide.

Mutants of human IL-2 (hIL-2) with decreased affinity to CD25 may forexample be generated by amino acid substitution at amino acid position35, 38, 42, 43, 45 or 72 or combinations thereof. Exemplary amino acidsubstitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L,R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D,F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R,Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K.Particular IL-2 mutants according to the invention comprise a mutationat an amino acid position corresponding to residue 42, 45, or 72 ofhuman IL-2, or a combination thereof. These mutants exhibitsubstantially similar binding affinity to the intermediate-affinity IL-2receptor, and have substantially reduced affinity to the α-subunit ofthe IL-2 receptor and the high-affinity IL-2 receptor compared to awild-type form of the IL-2 mutant.

Other characteristics of useful mutants may include the ability toinduce proliferation of IL-2 receptor-bearing T and/or NK cells, theability to induce IL-2 signaling in IL-2 receptor-bearing T and/or NKcells, the ability to generate interferon (IFN)-γ as a secondarycytokine by NK cells, a reduced ability to induce elaboration ofsecondary cytokines—particularly IL-10 and TNF-α—by peripheral bloodmononuclear cells (PBMCs), a reduced ability to activate regulatory Tcells, a reduced ability to induce apoptosis in T cells, and a reducedtoxicity profile in vivo.

In one embodiment according to the invention, the amino acid mutationthat abolishes or reduces affinity of the mutant IL-2 polypeptide to thehigh-affinity IL-2 receptor and preserves affinity of the mutant IL-2polypeptide to the intermediate-affinity IL-2 receptor is at a positioncorresponding to residue 72 of human IL-2. In one embodiment said aminoacid mutation is an amino acid substitution. In one embodiment saidamino acid substitution is selected from the group of L72G, L72A, L72S,L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a more specificembodiment said amino acid mutation is the amino acid substitution L72G.

In a particular aspect the invention provides a mutant IL-2 polypeptidecomprising a first and a second amino acid mutation that abolishes orreduces affinity of the mutant IL-2 polypeptide to the α-subunit of theIL-2 receptor and preserves affinity of the mutant IL-2 polypeptide tothe intermediate affinity IL-2 receptor. In one embodiment said firstamino acid mutation is at a position corresponding to residue 72 ofhuman IL-2. In one embodiment said first amino acid mutation is an aminoacid substitution. In a specific embodiment said first amino acidmutation is an amino acid substitution selected from the group of L72G,L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In an evenmore specific embodiment said amino acid substitution is L72G. Saidsecond amino acid mutation is at a different position than said firstamino acid mutation. In one embodiment said second amino acid mutationis at a position selected from a position corresponding to residue 35,38, 42, 43 and 45 of human IL-2. In one embodiment said second aminoacid mutation is an amino acid substitution. In a specific embodimentsaid amino acid substitution is selected from the group of K35E, K35A,R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G,F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S,Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. In a particular embodimentsaid second amino acid mutation is at a position corresponding toresidue 42 or 45 of human IL-2. In a specific embodiment said secondamino acid mutation is an amino acid substitution, selected from thegroup of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K,Y45A, Y450, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45C. In amore specific embodiment said second amino acid mutation is the aminoacid substitution F42A or Y45A. In a more particular embodiment saidsecond amino acid mutation is at the position corresponding to residue42 of human IL-2. In a specific embodiment said second amino acidmutation is an amino acid substitution, selected from the group of F42A,F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, and F42K. In a morespecific embodiment said amino acid substitution is F42A. In anotherembodiment said second amino acid mutation is at the positioncorresponding to residue 45 of human IL-2. In a specific embodiment saidsecond amino acid mutation is an amino acid substitution, selected fromthe group of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, andY45K. In a more specific embodiment said amino acid substitution isY45A. In certain embodiments the mutant IL-2 polypeptide comprises athird amino acid mutation that abolishes or reduces affinity of themutant IL-2 polypeptide to the α-subunit of the IL-2 receptor andpreserves affinity of the mutant IL-2 polypeptide to theintermediate-affinity IL-2 receptor, each compared to a wild-type IL-2polypeptide. Said third amino acid mutation is at a different positionthan said first and second amino acid mutations. In one embodiment saidthird amino acid mutation is at a position selected from a positioncorresponding to residue 35, 38, 42, 43 and 45 of human IL-2. In apreferred embodiment said third amino acid mutation is at a positioncorresponding to residue 42 or 45 of human IL-2. In one embodiment saidthird amino acid mutation is at a position corresponding to residue 42of human IL-2. In another embodiment said third amino acid mutation isat a position corresponding to residue 45 of human IL-2. In oneembodiment said third amino acid mutation is an amino acid substitution.In a specific embodiment said amino acid substitution is selected fromthe group of K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y,R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K,K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. Ina more specific embodiment said amino acid substitution is selected fromthe group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K,Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K. In aneven more specific embodiment said amino acid substitution is F42A orY45A. In one embodiment said amino acid substitution is F42A. In anotherembodiment said amino acid substitution is Y45A. In certain embodimentsthe mutant IL-2 polypeptide does not comprise an amino acid mutation atthe position corresponding to residue 38 of human IL-2.

In an even more particular aspect of the invention is provided a mutantIL-2 polypeptide comprising three amino acid mutations that abolish orreduce affinity of the mutant IL-2 polypeptide to the α-subunit of theIL-2 receptor but preserve affinity of the mutant IL-2 polypeptide tothe intermediate affinity IL-2 receptor. In one embodiment said threeamino acid mutations are at positions corresponding to residue 42, 45and 72 of human IL-2. In one embodiment said three amino acid mutationsare amino acid substitutions. In one embodiment said three amino acidmutations are amino acid substitutions selected from the group of F42A,F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S,Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q,L72E, L72N, L72D, L72R, and L72K. In a specific embodiment said threeamino acid mutations are amino acid substitutions F42A, Y45A and L72G.

In certain embodiments said amino acid mutation reduces the affinity ofthe mutant IL-2 polypeptide to the α-subunit of the IL-2 receptor by atleast 5-fold, specifically at least 10-fold, more specifically at least25-fold. In embodiments where there is more than one amino acid mutationthat reduces the affinity of the mutant IL-2 polypeptide to theα-subunit of the IL-2 receptor, the combination of these amino acidmutations may reduce the affinity of the mutant IL-2 polypeptide to theα-subunit of the IL-2 receptor by at least 30-fold, at least 50-fold, oreven at least 100-fold. In one embodiment said amino acid mutation orcombination of amino acid mutations abolishes the affinity of the mutantIL-2 polypeptide to the α-subunit of the IL-2 receptor so that nobinding is detectable by surface plasmon resonance as describedhereinbelow.

Substantially similar binding to the intermediate-affinity receptor,i.e. preservation of the affinity of the mutant IL-2 polypeptide to saidreceptor, is achieved when the IL-2 mutant exhibits greater than about70% of the affinity of a wild-type form of the IL-2 mutant to theintermediate-affinity IL-2 receptor. IL-2 mutants of the invention mayexhibit greater than about 80% and even greater than about 90% of suchaffinity.

The inventors have found that a reduction of the affinity of IL-2 forthe α-subunit of the IL-2 receptor in combination with elimination ofthe O-glycosylation of IL-2 results in an IL-2 protein with improvedproperties. For example, elimination of the O-glycosylation site resultsin a more homogenous product when the mutant IL-2 polypeptide isexpressed in mammalian cells such as CHO or HEK cells.

Thus, in certain embodiments the mutant IL-2 polypeptide according tothe invention comprises an additional amino acid mutation whicheliminates the O-glycosylation site of IL-2 at a position correspondingto residue 3 of human IL-2. In one embodiment said additional amino acidmutation which eliminates the O-glycosylation site of IL-2 at a positioncorresponding to residue 3 of human IL-2 is an amino acid substitution.Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D,T3R, T3K, and T3P. In a specific embodiment, said additional amino acidmutation is the amino acid substitution T3A.

In certain embodiments the mutant IL-2 polypeptide is essentially afull-length IL-2 molecule. In certain embodiments the mutant IL-2polypeptide is a human IL-2 molecule. In one embodiment the mutant IL-2polypeptide comprises the sequence of SEQ ID NO: 1 with at least oneamino acid mutation that abolishes or reduces affinity of the mutantIL-2 polypeptide to the α-subunit of the IL-2 receptor but preserveaffinity of the mutant IL-2 polypeptide to the intermediate affinityIL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 1without said mutation. In another embodiment, the mutant IL-2polypeptide comprises the sequence of SEQ ID NO: 3 with at least oneamino acid mutation that abolishes or reduces affinity of the mutantIL-2 polypeptide to the α-subunit of the IL-2 receptor but preserveaffinity of the mutant IL-2 polypeptide to the intermediate affinityIL-2 receptor, compared to an IG-2 polypeptide comprising SEQ ID NO: 3without said mutation.

In a specific embodiment, the mutant IL-2 polypeptide can elicit one ormore of the cellular responses selected from the group consisting of:proliferation in an activated T lymphocyte cell, differentiation in anactivated T lymphocyte cell, cytotoxic T cell (CTL) activity,proliferation in an activated B cell, differentiation in an activated Bcell, proliferation in a natural killer (NK) cell, differentiation in aNK cell, cytokine secretion by an activated T cell or an NK cell, andNK/lymphocyte activated killer (LAK) antitumor cytotoxicity.

In one embodiment the mutant IL-2 polypeptide has a reduced ability toinduce IL-2 signaling in regulatory T cells, compared to a wild-typeIL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide inducesless activation-induced cell death (AICD) in T cells, compared to awild-type IL-2 polypeptide. In one embodiment the mutant IL-2polypeptide has a reduced toxicity profile in vivo, compared to awild-type IL-2 polypeptide. In one embodiment the mutant IL-2polypeptide has a prolonged serum half-life, compared to a wild-typeIL-2 polypeptide.

A particular mutant IL-2 polypeptide according to the inventioncomprises four amino acid substitutions at positions corresponding toresidues 3, 42, 45 and 72 of human IL-2. Specific amino acidsubstitutions are T3A, F42A, Y45A and L72G. As demonstrated in theappended Examples, said quadruple mutant IL-2 polypeptide exhibits nodetectable binding to CD25, reduced ability to induce apoptosis in Tcells, reduced ability to induce IL-2 signaling in T_(reg) cells, and areduced toxicity profile in vivo. However, it retains ability toactivate IL-2 signaling in effector cells, to induce proliferation ofeffector cells, and to generate IFN-γ as a secondary cytokine by NKcells.

Moreover, said mutant IL-2 polypeptide has further advantageousproperties, such as reduced surface hydrophobicity, good stability, andgood expression yield, as described in the Examples. Unexpectedly, saidmutant IL-2 polypeptide also provides a prolonged serum half-life,compared to wild-type IL-2.

IL-2 mutants of the invention, in addition to having mutations in theregion of IL-2 that forms the interface of IL-2 with CD25 or theglycosylation site, also may have one or more mutations in the aminoacid sequence outside these regions. Such additional mutations in humanIL-2 may provide additional advantages such as increased expression orstability. For example, the cysteine at position 125 may be replacedwith a neutral amino acid such as serine, alanine, threonine or valine,yielding C125S IL-2, C125A IL-2, C125T IL-2 or C12SV IL-2 respectively,as described in U.S. Pat. No. 4,518,584. As described therein, one mayalso delete the N-terminal alanine residue of IL-2 yielding such mutantsas des-A1 C125S or des-A1 C125A. Alternatively or conjunctively, theIL-2 mutant may include a mutation whereby methionine normally occurringat position 104 of wild-type human IL-2 is replaced by a neutral aminoacid such as alanine (see U.S. Pat. No. 5,206,344). The resultingmutants, e. g., des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2,M104A C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (theseand other mutants may be found in U.S. Pat. No. 5,116,943 and in Weigeret al., Eur J Biochem 180, 295-300 (1989)) may be used in conjunctionwith the particular IL-2 mutations of the invention.

Thus, in certain embodiments the mutant IL-2 polypeptide according tothe invention comprises an additional amino acid mutation at a positioncorresponding to residue 125 of human IL-2. In one embodiment saidadditional amino acid mutation is the amino acid substitution C125A.

The skilled person will be able to determine which additional mutationsmay provide additional advantages for the purpose of the invention. Forexample, he will appreciate that amino acid mutations in the IL-2sequence that reduce or abolish the affinity of IL-2 to theintermediate-affinity IL-2 receptor, such as D20T, N88R or Q126D (seee.g. US 2007/0036752), may not be suitable to include in the mutant IL-2polypeptide according to the invention.

In one embodiment the mutant IL-2 polypeptide of the invention comprisesa sequence selected from the group of SEQ ID NO: 7, SEQ ID NO: 11, SEQID NO: 15, aid SEQ ID NO: 19. In a specific embodiment the mutant IL-2polypeptide of the invention comprises a sequence of SEQ ID NO: 15 orSEQ ID NO: 19. In an even more specific embodiment the mutant IL-2polypeptide comprises a sequence of SEQ ID NO: 19.

Mutant IL-2 polypeptides of the invention are particularly useful in thecontext of IL-2 fusion proteins such as IL-2 bearing immunoconjugates.Such fusion proteins comprise a mutant IL-2 polypeptide of the inventionfused to a non-IL-2 moiety. The non-IL-2 moiety can be a synthetic ornatural protein or a portion or variant thereof. Exemplary non-IL-2moieties include albumin, or antibody domains such as Fc domains orantigen binding domains of immunoglobulins.

IL-2 bearing immunoconjugates are fusion proteins comprising an antigenbinding moiety and an IL-2 moiety. They significantly increase theefficacy of IL-2 therapy by directly targeting IL-2 e.g. into a tumormicroenvironment. According to the invention, an antigen binding moietycan be a whole antibody or immunoglobulin, or a portion or variantthereof that has a biological function such as antigen specific bindingaffinity.

The benefits of immunoconjugate therapy are readily apparent. Forexample, an antigen binding moiety of an immunoconjugate recognizes atumor-specific epitope and results in targeting of the immunoconjugatemolecule to the tumor site. Therefore, high concentrations of IL-2 canbe delivered into the tumor microenvironment, thereby resulting inactivation and proliferation of a variety of immune effector cellsmentioned herein using a much lower dose of the immunoconjugate thanwould be required for unconjugated IL-2. Moreover, since application ofIL-2 in form of immunoconjugates allows lower doses of the cytokineitself, the potential for undesirable side effects of IL-2 isrestricted, and targeting the IL-2 to a specific site in the body bymeans of an immunoconjugate may also result in a reduction of systemicexposure and thus less side effects than obtained with unconjugatedIL-2. In addition, the increased circulating half-life of animmunoconjugate compared to unconjugated IL-2 contributes to theefficacy of the immunoconjugate. However, this characteristic of IL-2immunoconjugates may again aggravate potential side effects of the IL-2molecule: Because of the significantly longer circulating half-life ofIL-2 immunoconjugate in the bloodstream relative to unconjugated IL-2,the probability for IL-2 or other portions of the fusion proteinmolecule to activate components generally present in the vasculature isincreased. The same concern applies to other fusion proteins thatcontain IL-2 fused to another moiety such as Fc or albumin, resulting inan extended half-life of IL-2 in the circulation. Therefore animmunoconjugate comprising a mutant IL-2 polypeptide according to theinvention, with reduced toxicity compared to wild-type forms of IL-2, isparticularly advantageous.

Accordingly, the invention further provides a mutant IL-2 polypeptide asdescribed hereinbefore, linked to at least one non-IL-2 moiety. In oneembodiment the mutant IL-2 polypeptide and the non-IL-2 moiety form afusion protein, i.e. the mutant IL-2 polypeptide shares a peptide bondwith the non-IL-2 moiety. In one embodiment the mutant IL-2 polypeptideis linked to a first and a second non-IL-2 moiety. In one embodiment themutant IL-2 polypeptide shares an amino- or carboxy-terminal peptidebond with the first antigen binding moiety, and the second antigenbinding moiety shares an amino- or carboxy-terminal peptide bond witheither i) the mutant IL-2 polypeptide or ii) the first antigen bindingmoiety. In a specific embodiment the mutant IL-2 polypeptide shares acarboxy-terminal peptide bond with said first non-IL-2 moiety and anamino-terminal peptide bond with said second non-IL-2 moiety. In oneembodiment said non-IL-2 moiety is a targeting moiety. In a particularembodiment said non-IL-2 moiety is an antigen binding moiety (thusforming an immunoconjugate with the mutant IL-2 polypeptide, asdescribed in more detail hereinbelow). In certain embodiments theantigen binding moiety is an antibody or an antibody fragment. In oneembodiment the antigen binding moiety is a full-length antibody. In oneembodiment the antigen binding moiety is an immunoglobulin molecule,particularly an IgG class immunoglobulin molecule, more particularly anIgG₁ subclass immunoglobulin molecule. In one such embodiment, themutant IL-2 polypeptide shares an amino-terminal peptide bond with oneof the immunoglobulin heavy chains. In another embodiment the antigenbinding moiety is an antibody fragment. In some embodiments said antigenbinding moiety comprises an antigen binding domain of an antibodycomprising an antibody heavy chain variable region and an antibody lightchain variable region. In a more specific embodiment the antigen bindingmoiety is a Fab molecule or a scFv molecule. In a particular embodimentthe antigen binding moiety is a Fab molecule. In another embodiment theantigen binding moiety is a scFv molecule. In one embodiment saidantigen binding moiety is directed to an antigen presented on a tumorcell or in a tumor cell environment. In a preferred embodiment saidantigen is selected from the group of Fibroblast Activation Protein(FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C(TNC A2), the Extra Domain B of Fibronectin (EDB), CarcinoembryonicAntigen (CEA) and the Melanoma-associated Chondroitin SulfateProteoglycan (MCSP). Where the mutant IL-2 polypeptide is linked to morethan one antigen binding moiety, e.g. a first and a second antigenbinding moiety, each antigen binding moiety can be independentlyselected from various forms of antibodies and antibody fragments. Forexample, the first antigen binding moiety can be a Fab molecule and thesecond antigen binding moiety can be a scFv molecule. In a specificembodiment each of said first and said second antigen binding moietiesis a scFv molecule or each of said first and said second antigen bindingmoieties is a Fab molecule. In a particular embodiment each of saidfirst and said second antigen binding moieties is a Fab molecule.Likewise, where the mutant IL-2 polypeptide is linked to more than oneantigen binding moiety, e.g. a first and a second antigen bindingmoiety, the antigen to which each of the antigen binding moieties isdirected can be independently selected. In one embodiment said first andsaid second antigen binding moieties are directed to different antigens.In another embodiment said first and said second antigen bindingmoieties are directed to the same antigen. As described above, theantigen is particularly an antigen presented on a tumor cell or in atumor cell environment, more particularly an antigen selected from thegroup of Fibroblast Activation Protein (FAP), the A1 domain ofTenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the ExtraDomain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) and theMelanoma-associated Chondroitin Sulfate Proteoglycan (MCSP). The antigenbinding region may further incorporate any of the features, singly or incombination, described herein in relation to antigen binding domains ofimmunoconjugates.

Immunoconjugates

In a particular aspect the invention provides an immunoconjugatecomprising a mutant IL-2 polypeptide comprising one or more amino acidmutation that abolishes or reduces affinity of the mutant IL-2polypeptide to the α-subunit of the IL-2 receptor and preserves affinityof the mutant IL-2 polypeptide to the intermediate-affinity IL-2receptor, and at least one antigen binding moiety. In one embodimentaccording to the invention, the amino acid mutation that abolishes orreduces affinity of the mutant IL-2 polypeptide to the α-subunit of theIL-2 receptor and preserves affinity of the mutant IL-2 polypeptide tothe intermediate affinity IL-2 receptor is at a position selected from aposition corresponding to residue 42, 45 and 72 of human IL-2. In oneembodiment said amino acid mutation is an amino acid substitution. Inone embodiment said amino acid mutation is an amino acid substitutionselected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N,F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R,Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K,more specifically an amino acid substitution selected from the group ofF42A, Y45A and L72G. In one embodiment the amino acid mutation is at aposition corresponding to residue 42 of human IL-2. In a specificembodiment said amino acid mutation is an amino acid substitutionselected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N,F42D, F42R, and F42. In an even more specific embodiment said amino acidsubstitution is F42A. In another embodiment the amino acid mutation isat a position corresponding to residue 45 of human IL-2. In a specificembodiment said amino acid mutation is an amino acid substitutionselected from the group of Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N,Y45D, Y45R, and Y45K. In an even more specific embodiment said aminoacid substitution is Y45A. In yet another embodiment the amino acidmutation is at a position corresponding to residue 72 of human IL-2. Ina specific embodiment said amino acid mutation is an amino acidsubstitution selected from the group of L72G, L72A, L72S, L72T, L72Q,L72E, L72N, L72D, L72R, and L72K. In an even more specific embodimentsaid amino acid substitution is L72G. In certain embodiments, the mutantIL-2 polypeptide according to the invention does not comprise an aminoacid mutation at a position corresponding to residue 38 of human IL-2.In a particular embodiment, the mutant IL-2 polypeptide comprised in theimmunoconjugate of the invention comprises at least a first and a secondamino acid mutation that abolishes or reduces affinity of the mutantIL-2 polypeptide to the α-subunit of the IL-2 receptor and preservesaffinity of the mutant IL-2 polypeptide to the intermediate affinityIL-2 receptor. In one embodiment said first and second amino acidmutations are at two positions selected from the positions correspondingto residue 42, 45 and 72 of human IL-2. In one embodiment said first andsecond amino acid mutations are amino acid substitutions. In oneembodiment said first and second amino acid mutations are amino acidsubstitutions selected from the group of F42A, F42G, F42S, F42T, F42Q,F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N,Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R,and L72K. In a particular embodiment said first and second amino acidmutations are amino acid substitutions selected from the group of F42A,Y45A and L72G. The mutant IL-2 polypeptide may further incorporate anyof the features, singly or in combination, described in the precedingparagraphs in relation to the mutant IL-2 polypeptides of the invention.In one embodiment said mutant IL-2 polypeptide shares an amino- orcarboxy-terminal peptide bond with said antigen binding moiety comprisedin the immunoconjugate, i.e. the immunoconjugate is a fusion protein. Incertain embodiments said antigen binding moiety is an antibody or anantibody fragment. In some embodiments said antigen binding moietycomprises an antigen binding domain of an antibody comprising anantibody heavy chain variable region and an antibody light chainvariable region. The antigen binding region may incorporate any of thefeatures, singly or in combination, described hereinabove or below inrelation to antigen binding domains.

Immunoconjugate Formats

Particularly suitable immunoconjugate formats are described in PCTpublication no. WO 2011/020783, which is incorporated herein byreference in its entirety. These immunoconjugates comprise at least twoantigen binding domains. Thus, in one embodiment, the immunoconjugateaccording to the present invention comprises at least a first mutantIL-2 polypeptide as described herein, and at least a first and a secondantigen binding moiety. In a particular embodiment, said first andsecond antigen binding moiety are independently selected from the groupconsisting of an Fv molecule, particularly a scFv molecule, and a Fabmolecule. In a specific embodiment, said first mutant IL-2 polypeptideshares an amino- or carboxy-terminal peptide bond with said firstantigen binding moiety and said second antigen binding moiety shares anamino- or carboxy-terminal peptide bond with either i) the first mutantIL-2 polypeptide or ii) the first antigen binding moiety. In aparticular embodiment, the immunoconjugate consists essentially of afirst mutant IL-2 polypeptide and first and second antigen bindingmoieties, joined by one or more linker sequences. Such formats have theadvantage that they bind with high affinity to the target antigen (suchas a tumor antigen), but only monomeric binding to the IL-2 receptor,thus avoiding targeting the immunoconjugate to IL-2 receptor bearingimmune cells at other locations than the target site. In a particularembodiment, a first mutant IL-2 polypeptide shares a carboxy-terminalpeptide bond with a first antigen binding moiety and further shares anamino-terminal peptide bond with a second antigen binding moiety. Inanother embodiment, a first antigen binding moiety shares acarboxy-terminal peptide bond with a first mutant IL-2 polypeptide, andfurther shares an amino-terminal peptide bond with a second antigenbinding moiety. In another embodiment, a first antigen binding moietyshares an amino-terminal peptide bond with a first mutant IL-2polypeptide, and further shares a carboxy-terminal peptide with a secondantigen binding moiety. In a particular embodiment, a mutant IL-2polypeptide shares a carboxy-terminal peptide bond with a first heavychain variable region and further shares an amino-terminal peptide bondwith a second heavy chain variable region. In another embodiment amutant IL-2 polypeptide shares a carboxy-terminal peptide bond with afirst light chain variable region and further shares an amino-terminalpeptide bond with a second light chain variable region. In anotherembodiment, a first heavy or light chain variable region is joined by acarboxy-terminal peptide bond to a first mutant IL-2 polypeptide and isfurther joined by an amino-terminal peptide bond to a second heavy orlight chain variable region. In another embodiment, a first heavy orlight chain variable region is joined by an amino-terminal peptide bondto a first mutant IL-2 polypeptide and is further joined by acarboxy-terminal peptide bond to a second heavy or light chain variableregion. In one embodiment, a mutant IL-2 polypeptide shares acarboxy-terminal peptide bond with a first Fab heavy or light chain andfurther shares an amino-terminal peptide bond with a second Fab heavy orlight chain. In another embodiment, a first Fab heavy or light chainshares a carboxy-terminal peptide bond with a first mutant IL-2polypeptide and further shares an amino-terminal peptide bond with asecond Fab heavy or light chain. In other embodiments, a first Fab heavyor light chain shares an amino-terminal peptide bond with a first mutantIL-2 polypeptide and further shares a carboxy-terminal peptide bond witha second Fab heavy or light chain. In one embodiment, theimmunoconjugate comprises at least a first mutant IL-2 polypeptidesharing an amino-terminal peptide bond with one or more scFv moleculesand further sharing a carboxy-terminal peptide bond with one or morescFv molecules.

Other particularly suitable immunoconjugate formats comprise animmunoglobulin molecule as antigen binding moiety. In one suchembodiment, the immunoconjugate comprises at least one mutant IL-2polypeptide as described herein and an immunoglobulin molecule,particularly an IgG molecule, more particularly an IgG₁ molecule. In oneembodiment the immunoconjugate comprises not more than one mutant IL-2polypeptide. In one embodiment the immunoglobulin molecule is human. Inone embodiment the mutant IL-2 polypeptide shares an amino- orcarboxy-terminal peptide bond with the immunoglobulin molecule. In oneembodiment, the immunoconjugate essentially consists of a mutant IL-2polypeptide and an immunoglobulin molecule, particularly an IgGmolecule, more particularly an IgG₁ molecule, joined by one or morelinker sequences. In a specific embodiment the mutant IL-2 polypeptideis joined at its amino-terminal amino acid to the carboxy-terminal aminoacid of one of the immunoglobulin heavy chains. In certain embodiments,the immunoglobulin molecule comprises in the Fc domain a modificationpromoting heterodimerization of two non-identical immunoglobulin heavychains. The site of most extensive protein-protein interaction betweenthe two polypeptide chains of a human IgG Fc domain is in the CH3 domainof the Fc domain. Thus, in one embodiment said modification is in theCH3 domain of the Fc domain. In a specific embodiment said modificationis a knob-into-hole modification, comprising a knob modification in oneof the immunoglobulin heavy chains and a hole modification in the otherone of the immunoglobulin heavy chains. The knob-into-hole technology isdescribed e.g. in U.S. Pat. Nos. 5,731,168; 7,695,936; Ridgway et al.,Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).Generally, the method involves introducing a protuberance (“knob”) atthe interface of a first polypeptide and a corresponding cavity (“hole”)in the interface of a second polypeptide, such that the protuberance canbe positioned in the cavity so as to promote heterodimer formation andhinder homodimer formation. Protuberances are constructed by replacingsmall amino acid side chains from the interface of the first polypeptidewith larger side chains (e.g. tyrosine or tryptophan). Compensatorycavities of identical or similar size to the protuberances are createdin the interface of the second polypeptide by replacing large amino acidside chains with smaller ones (e.g. alanine or threonine). Theprotuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis. In a specific embodiment a knob modificationcomprises the amino acid substitution T366W in one of the twoimmunoglobulin heavy chains, and the hole modification comprises theamino acid substitutions T366S, L368A and Y407V in the other one of thetwo immunoglobulin heavy chains. In a further specific embodiment,immunoglobulin heavy chain comprising the knob modification additionallycomprises the amino acid substitution S354C, and the immunoglobulinheavy chain comprising the hole modification additionally comprises theamino acid substitution Y349C. Introduction of these two cysteineresidues results in formation of a disulfide bridge between thetwo-heavy chains, further stabilizing the dimer (Carter, J ImmunolMethods 248, 7-(2001)).

In a particular embodiment the mutant IL-2 polypeptide is joined to thecarboxy-terminal amino acid of the immunoglobulin heavy chain comprisingthe knob modification.

In an alternative embodiment a modification promoting heterodimerizationof two non-identical polypeptide chains comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the twopolypeptide chains by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

An Fc domain confers to the immunoconjugate favorable pharmacokineticproperties, including a long serum half-life which contributes to goodaccumulation in the target tissue and a favorable tissue-blooddistribution ratio. At the same time it may, however, lead toundesirable targeting of the immunoconjugate to cells expressing Fcreceptors rather than to the preferred antigen-bearing cells. Moreover,the co-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the IL-2 polypeptide and the longhalf-life of the immunoconjugate, results in excessive activation ofcytokine receptors and severe side effects upon systemic administration.In line with this, conventional IgG-IL-2 immunoconjugates have beendescribed to be associated with infusion reactions (see e.g. King etal., J Clin Oncol 22, 4463-4473 (2004)).

Accordingly, in certain embodiments the immunoglobulin moleculecomprised in the immunoconjugate according to the invention isengineered to have reduced binding affinity to an Fc receptor. In onesuch embodiment the immunoglobulin comprises in its Fc domain one ormore amino acid mutation that reduces the binding affinity of theimmunoconjugate to an Fc receptor. Typically, the same one or more aminoacid mutation is present in each of the two immunoglobulin heavy chains.In one embodiment said amino acid mutation reduces the binding affinityof the immunoconjugate to the Fc receptor by at least 2-fold, at least5-fold, or at least 10-fold. In embodiments where there is more than oneamino acid mutation that reduces the binding affinity of theimmunoconjugate to the Fc receptor, the combination of these amino acidmutations may reduce the binding affinity of the Fc domain to the Fcreceptor by at least 10-fold, at least 20-fold, or even at least50-fold. In one-embodiment the immunoconjugate comprising an engineeredimmunoglobulin molecule exhibits less than 20%, particularly less than10%, more particularly less than 5% of the binding affinity to an Fcreceptor as compared to an immunoconjugate comprising a non-engineeredimmunoglobulin molecule. In one embodiment the Fc receptor is anactivating Fc receptor. In a specific embodiment the Fc receptor is anFcγ receptor, more specifically an FcγRIa, FcγRI or FcγRIa receptor.Preferably, binding to each of these receptors is reduced. In someembodiments binding affinity to a complement component, specificallybinding affinity to C1q, is also reduced. In one embodiment bindingaffinity to neonatal Fc receptor (FcRn) is not reduced. Substantiallysimilar binding to FcRn, i.e. preservation of the binding affinity ofthe immunoglobulin to said receptor, is achieved when the immunoglobulin(or the immunoconjugate comprising said immunoglobulin) exhibits greaterthan about 70% of the binding affinity of a non-engineered form of theimmunoglobulin (or the immunoconjugate comprising said non-engineeredform of the immunoglobulin) to FcRn. Immunoglobulins, orimmunoconjugates comprising said immunoglobulins, may exhibit greaterthan about 80% and even greater than about 90% of such affinity. In oneembodiment the amino acid mutation is an amino acid substitution. In oneembodiment the immunoglobulin comprises an amino acid substitution atposition P329 of the immunoglobulin heavy chain (Kabat numbering). In amore specific embodiment the amino acid substitution is P329A or P329G,particularly P3290. In one embodiment the immunoglobulin comprises afurther amino acid substitution at a position selected from S228, E233,L234, L235, N297 and P331 of the immunoglobulin heavy chain. In a morespecific embodiment the further amino acid substitution is S228P, E233P,L234A, L235A, L235E, N297A, N297D or P331 S. In a particular embodimentthe immunoglobulin comprises amino acid substitutions at positions P329,L234 and L235 of the immunoglobulin heavy chain. In a more particularembodiment the immunoglobulin comprises the amino acid mutations L234A,L235A and P329G (LALA P329G). This combination of amino acidsubstitutions almost completely abolishes Fcγ receptor binding of ahuman IgG molecule, and hence decreases effector function includingantibody-dependent cell-mediated cytotoxicity (ADCC).

In certain embodiments, the immunoconjugate comprises one or moreproteolytic cleavage sites located between mutant IL-2 polypeptide andantigen binding moieties.

Components of the immunoconjugate (e.g. antigen binding moieties and/ormutant IL-2 polypeptide) may be linked directly or through variouslinkers, particularly peptide linkers comprising one or more aminoacids, typically about 2-20 amino acids, that are described herein orare known in the art. Suitable, non-immunogenic linker peptides include,for example, (G4S)_(n), (SG₄)_(n) or G₄(SG₄)_(n) linker peptides,wherein n is generally a number between 1 and 10, typically between 2and 4:

Antigen Binding Moieties

The antigen binding moiety of the immunoconjugate of the invention isgenerally a polypeptide molecule that binds to a specific antigenicdeterminant and is able to direct the entity to which it is attached(e.g. a mutant IL-2 polypeptide or a second antigen binding moiety) to atarget site, for example to a specific type of tumor cell or tumorstroma that bears the antigenic determinant. The immunoconjugate canbind to antigenic determinants found, for example, on the surfaces oftumor cells, on the surfaces of virus-infected cells, on the surfaces ofother diseased cells, free in blood serum, and/or in the extracellularmatrix (ECM).

Non-limiting examples of tumor antigens include MAGE, MART-1/Melan-A,gp100, Dipeptidyl peptidage IV (DPPIV), adenosine deaminase-bindingprotein (ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenicepitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA)and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family oftumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin,α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME,NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin,Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viralproducts such as human papilloma virus proteins, Smad family of tumorantigens, Imp-1, PIA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,SCP-1 and CT-7, and c-erbB-2.

Non-limiting examples of viral antigens include influenza virushemagglutinin, Epstein-Banrr virus LMP-1, hepatitis C virus E2glycoprotein, HIV gp160, and HIV gp120.

Non-limiting examples of ECM antigens include syndecan, heparanase,integrins, osteopontin, link, cadherins, laminin; laminin type EGF,lectin, fibronectin, notch, tenascin, and matrixin.

The immunoconjugates of the invention can bind to the following specificnon-limiting examples of cell surface antigens: FAP, Her2, EGFR, IGF-1R,CD2 (T-cell surface antigen), CD3 (heteromultimer associated with theTCR), CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD30(cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumornecrosis factor receptor), IL-6R-(IL6 receptor), CD20, MCSP, and PDGFPR(β platelet-derived growth factor receptor).

In one embodiment, the immunoconjugate of the invention comprises two ormore antigen binding moieties, wherein each of these antigen bindingmoieties specifically binds to the same antigenic determinant. Inanother embodiment, the immunoconjugate of the invention comprises twoor more antigen binding moieties, wherein each of these antigen bindingmoieties specifically binds to different antigenic determinants.

The antigen binding moiety can be any type of antibody or fragmentthereof that retains specific binding to an antigenic determinant.Antibody fragments include, but are not limited to, V_(H) fragments,V_(L) fragments, Fab fragments, F(ab′)₂ fragments, scFv fragments, Fvfragments, minibodies, diabodies, triabodies, and tetrabodies (see e.g.Hudson and Souriau, Nature Mod 9, 129-134 (2003)).

Particularly suitable antigen binding moieties are described in PCTpublication no. WO 2011/020783, which is incorporated herein byreference in its entirety.

In one embodiment, the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for the ExtraDomain B of fibronectin (EDB). In another embodiment, theimmunoconjugate comprises at least one, typically two or more antigenbinding moieties that can compete with monoclonal antibody L19 forbinding to an epitope of EDB. See, e.g., PCT publication WO 2007/128563A1 (incorporated herein by reference in its entirety). In yet anotherembodiment, the immunoconjugate comprises a polypeptide sequence whereina first Fab heavy chain derived from the L19 monoclonal antibody sharesa carboxy-terminal peptide bond with a mutant IL-2 polypeptide which inturn shares a carboxy-terminal peptide bond with a second Fab heavychain derived from the L19 monoclonal antibody. In yet anotherembodiment, the immunoconjugate comprises a polypeptide sequence whereina first Fab light chain derived from the L19 monoclonal antibody sharesa carboxy-terminal peptide bond with a mutant IL-2 polypeptide which inturn shares a carboxy-terminal peptide bond with a second Fab lightchain derived from the L19 monoclonal antibody. In a further embodiment,the immunoconjugate comprises a polypeptide sequence wherein a firstscFv derived from the L19 monoclonal antibody shares a carboxy-terminalpeptide bond with a mutant IL-2 polypeptide which in turn shares acarboxy-terminal peptide bond with a second scFv derived from the L19monoclonal antibody.

In a more specific embodiment, the immunoconjugate comprises thepolypeptide sequence of SEQ ID NO: 199 or a variant thereof that retainsfunctionality. In another embodiment, the immunoconjugate comprises aFab light chain derived from the L19 monoclonal antibody. In a morespecific embodiment, the immunoconjugate comprises a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 201 or a variant thereof that retainsfunctionality. In yet another embodiment, the immunoconjugate comprisestwo polypeptide sequences that are at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 199 and SEQ ID NO:201 or variants thereof that retain functionality. In another specificembodiment, the polypeptides are covalently linked, e.g., by a disulfidebond.

In one embodiment, the immunoconjugate of the invention comprises atleast one, typically two or more antigen binding moieties that arespecific for the A1 domain of Tenascin (TNC-A1). In another embodiment,the immunoconjugate comprises at least one, typically two or moreantigen binding moieties that can compete with monoclonal antibody F16for binding to an epitope of TNC-A1. See, e.g., PCT Publication WO2007/128563 A1 (incorporated herein by reference in its entirety). Inone embodiment, the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for the A1 and/orthe A4 domain of Tenascin (TNC-A1 or TNC-A4 or TNC-A1/A4). In anotherembodiment, the immunoconjugate comprises a polypeptide sequence whereina first Fab heavy chain specific for the A1 domain of Tenascin shares acarboxy-terminal peptide bond with a mutant IL-2 polypeptide, which inturn shares a carboxy-terminal peptide bond with a second Fab heavychain specific for the A1 domain of Tenascin. In yet another embodiment,the immunoconjugate comprises a polypeptide sequence wherein a first Fablight chain specific for the A1 domain of Tenascin shares acarboxy-terminal peptide bond with a mutant IL-2 polypeptide which inturn shares a carboxy-terminal peptide bond with a second Fab lightchain specific for the A1 domain of Tenascin. In a further embodiment,the immunoconjugate comprises a polypeptide sequence wherein a firstscFv specific for the A1 domain of Tenascin shares a carboxy-terminalpeptide bond with a mutant IL-2 polypeptide which in turn shares acarboxy-terminal peptide bond with a second scFv specific for the A1domain of Tenascin. In another embodiment, the immunoconjugate comprisesa polypeptide sequence wherein an immunoglobulin heavy chain specificfor TNC-A1 shares a carboxy-terminal peptide bond with a mutant IL-2polypeptide.

In a specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto either SEQ ID NO: 33 or SEQ ID NO: 35, or variants thereof thatretain functionality. In another specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to either SEQ ID NO: 29 or SEQ ID NO: 31, orvariants thereof that retain functionality. In a more specificembodiment, the antigen binding moieties of the immunoconjugate comprisea heavy chain variable region sequence that is at least about 80%, 85%;90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 33or SEQ ID NO: 35 or variants thereof that retain functionality, and alight chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 29or SEQ ID NO: 31 or variants thereof that retain functionality.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to either SEQ ID NO: 34 or SEQ ID NO: 36. Inyet another specific embodiment, the heavy chain variable regionsequence of the antigen-binding moieties of the immunoconjugate isencoded by the polynucleotide sequence of either SEQ ID NO: 34 or SEQ IDNO: 36. In another specific embodiment, the light chain variable regionsequence of the antigen binding moieties of the immunoconjugate isencoded by a polynucleotide sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 30 or SEQID NO: 32. In yet another specific embodiment, the light chain variableregion sequence of the antigen binding moieties of the immunoconjugateis encoded by the polynucleotide sequence of either SEQ ID NO: 30 or SEQID NO: 32.

In a specific embodiment, the immunoconjugate comprises a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 203 or variants thereof that retainfunctionality. In another specific embodiment, the immunoconjugate ofthe invention comprises a polypeptide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQID NO: 205 or SEQ ID NO: 215, or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the invention comprises a polypeptide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQID NO: 207 or SEQ ID NO: 237 or variants thereof that retainfunctionality. In a more specific embodiment, the immunoconjugate of thepresent invention comprises two polypeptide sequences that are at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 205 and SEQ ID NO: 207 or variants thereof that retainfunctionality. In another specific embodiment, the immunoconjugate ofthe present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 215 and SEQ ID NO: 237 of variants thereof that retainfunctionality.

In a specific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 204.In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by the polynucleotide sequence of SEQ IDNO: 204. In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toeither SEQ ID NO: 206 or SEQ ID NO: 216. In yet another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby the polynucleotide sequence of either SEQ ID NO: 206 or SEQ ID NO:216. In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical toeither SEQ ID NO: 208 or SEQ ID NO: 238. In yet another embodiment, theimmunoconjugate comprises a polypeptide sequence encoded by thepolynucleotide sequence of either SEQ ID NO: 208 or SEQ ID NO: 238.

In one embodiment, the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for the A2 domainof Tenascin (TNC-A2). In another embodiment, the immunoconjugatecomprises a polypeptide sequence wherein a first Fab heavy chainspecific for the A2 domain of Tenascin shares a carboxy-terminal peptidebond with a IL mutant IL-2 polypeptide, which in turn shares acarboxy-terminal peptide bond with a second Fab heavy chain specific forthe A2 domain of Tenascin. In yet another embodiment, theimmunoconjugate comprises a polypeptide sequence wherein a first Fablight chain specific for the A2 domain of Tenascin shares acarboxy-terminal peptide bond with a mutant IL-2 polypeptide, which inturn shares a carboxy-terminal peptide bond with a second Fab lightchain specific for the A2 domain of Tenascin. In another embodiment, theimmunoconjugate comprises a polypeptide sequence wherein animmunoglobulin heavy chain specific for TNC-A2 shares a carboxy-terminalpeptide bond with a mutant IL-2 polypeptide.

In a specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a sequence selected from the group of SEQ ID NO: 27, SEQ ID NO: 159,SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO:175, SEQ IDNO: 179, SEQ ID NO: 183 and SEQ ID NO: 187, or variants thereof thatretain functionality. In another specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to a sequence selected from the group of SEQID NO: 23, SEQ ID NO: 25; SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO:165,SEQ ID NO: 169, SEQ ID NO: 173, SEQ ID NO: 177, SEQ ID NO: 181 and SEQID NO: 185, or variants thereof that retain functionality. In a morespecific embodiment, the antigen binding moieties of the immunoconjugatecomprise a heavy chain variable region sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequenceselected from the group of SEQ ID NO: 27, SEQ ID NO: 159, SEQ ID NO:163, SEQ ID NO: 167, SEQ ID NO: 171, SEQ ID NO:175, SEQ ID NO: 179, SEQID NO: 183 and SEQ ID NO: 187, or variants thereof that retainfunctionality, and a light chain variable region sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toa sequence selected from the group of SEQ-ID NO: 23, SEQ ID NO: 25; SEQID NO: 157, SEQ ID NO: 161, SEQ ID NO:165, SEQ ID NO: 169, SEQ ID NO:173, SEQ ID NO: 177, SEQ ID NO: 181 and SEQ ID NO: 185, or variantsthereof that retain functionality.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to a sequence selected from the group of SEQID NO: 28, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO:172, SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184 and SEQ ID NO: 188.In yet another specific embodiment, the heavy chain variable regionsequence of the antigen binding moieties of the immunoconjugate isencoded by a polynucleotide sequence selected from the group of SEQ IDNO: 28, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172,SEQ ID NO: 176, SEQ ID NO: 180, SEQ ID NO: 184 and SEQ ID NO: 188. Inanother specific embodiment, the light chain variable region sequence ofthe antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to a sequence selected from the group of SEQID NO: 24, SEQ ID NO: 26, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO:166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQ ID NO: 182 andSEQ ID NO: 186. In yet another specific embodiment, the light chainvariable region sequence of the antigen binding moieties of theimmunoconjugate is encoded by a polynucleotide sequence selected fromthe group of of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 158, SEQ ID NO:162, SEQ ID NO: 166, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 178, SEQID NO: 182 and SEQ ID NO: 186.

In a specific embodiment, the immunoconjugate of the invention comprisesa polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the group ofSEQ ID NO: 241, SEQ ID NO: 243 and SEQ ID NO: 245, or variants thereofthat retain functionality. In another specific embodiment, theimmunoconjugate of the invention comprises a polypeptide sequence thatis at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to a sequence selected from the group of SEQ ID NO: 247, SEQID NO: 249 and SEQ ID NO: 251, or variants thereof that retainfunctionality. In a more specific embodiment, the immunoconjugate of thepresent invention comprises a polypeptide sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to asequence selected from the group of SEQ ID NO: 241, SEQ ID NO: 243, andSEQ ID NO: 245 or variants thereof that retain functionality, and apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the group ofSEQ ID NO: 247, SEQ ID NO: 249 and SEQ ID NO: 251 or variants thereofthat retain functionality. In another specific embodiment, theimmunoconjugate of the present invention comprises two polypeptidesequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 241 and either SEQ ID NO: 249 or SEQ IDNO: 251, or variants thereof that retain functionality. In yet anotherspecific embodiment, the immunoconjugate of the present inventioncomprises two polypeptide sequences that are at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 243 andeither SEQ ID NO: 247 or SEQ ID NO: 249, or variants thereof that retainfunctionality. In another specific embodiment, the immunoconjugate ofthe present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 245 and SEQ ID NO: 247, or variants thereof that retainfunctionality.

In a specific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequenceselected from the group of SEQ ID NO: 242, SEQ ID NO: 244 and SEQ ID NO:246. In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence selected fromthe group of of SEQ ID NO: 242, SEQ ID NO: 244 and SEQ ID NO: 246. Inanother specific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequenceselected from the group of SEQ ID NO: 248, SEQ ID NO: 250 and SEQ ID NO:252. In yet another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence selected fromthe group of of SEQ ID NO: 248, SEQ ID NO: 250 and SEQ ID NO: 252.

In one embodiment, the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for theFibroblast Activated Protein (FAP). In another embodiment, theimmunoconjugate comprises a polypeptide sequence wherein a first Fabheavy chain specific for FAP shares a carboxy-terminal peptide bond witha mutant IL-2 polypeptide, which in turn shares a carboxy-terminalpeptide bond with a second Fab heavy chain specific for FAP. In yetanother embodiment, the immunoconjugate comprises a polypeptide sequencewherein a first Fab light chain specific for FAP shares acarboxy-terminal peptide bond with a mutant IL-2 polypeptide, which inturn shares a carboxy-terminal peptide bond with a second Fab lightchain specific for FAP. In another embodiment, the immunoconjugatecomprises a polypeptide sequence wherein an immunoglobulin heavy chainspecific for FAP shares a carboxy-terminal peptide bond with a mutantIL-2 polypeptide.

In a specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a sequence selected from the group consisting of SEQ ID NO: 41, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59,SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO:79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ IDNO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115,SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ IDNO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151and SEQ ID NO: 155, or variants thereof that retain functionality. Inanother specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a light chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a sequence selected from the group consisting of: SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57,SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO:77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ IDNO: 97, SEQ ID NO: 101, SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113,SEQ ID NO: 117, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ IDNO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149and SEQ ID NO: 153, or variants thereof that retain functionality. In amore specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a sequence selected from the group consisting of SEQ ID NO: 41, SEQID NO: 45, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59,SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO:79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 95, SEQ IDNO: 99, SEQ ID NO: 103, SEQ ID NO: 107, SEQ ID NO: 111, SEQ ID NO: 115,SEQ ID NO: 119, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ IDNO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151and SEQ ID NO: 155, or variants thereof that retain functionality, and alight chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selectedfrom the group consisting of: SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO:43, SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ IDNO: 65, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 101,SEQ ID NO: 105, SEQ ID NO: 109, SEQ ID NO: 113, SEQ ID NO: 117, SEQ IDNO: 121, SEQ ID NO: 125, SEQ ID NO. 129, SEQ ID NO: 133, SEQ ID NO: 137,SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149 and SEQ ID NO: 153, orvariants thereof that retain functionality. In one embodiment, antigenbinding moieties of the immunoconjugate comprise the heavy chainvariable region sequence of SEQ ID NO: 41 and the light chain variableregion sequence of SEQ ID NO: 39. In one embodiment, antigen bindingmoieties of the immunoconjugate comprise the heavy chain variable regionsequence of SEQ ID NO: 51 and the light chain variable region sequenceof SEQ ID NO: 49. In one embodiment, antigen binding moieties of theimmunoconjugate comprise the heavy chain variable region sequence of SEQID NO: 111 and the light chain variable region sequence of SEQ ID. NO:109. In one embodiment, antigen binding moieties of the immunoconjugatecomprise the heavy chain variable region sequence of SEQ ID NO: 143 andthe light chain variable region sequence of SEQ ID NO: 141. In oneembodiment, antigen binding moieties of the immunoconjugate comprise theheavy chain variable region sequence of SEQ ID NO: 151 and the lightchain variable region sequence of SEQ ID NO: 149.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to a sequence selected from the groupconsisting of: SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO:52, SEQ ID NO: 56, SEQ ID NO. 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ IDNO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQID NO: 92, SEQ ID NO: 96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO:108, SEQ ID NO: 112, SEQ ID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO:144, SEQ ID NO: 148, SEQ ID NO: 152, and SEQ ID NO: 156. In yet anotherspecific embodiment, the heavy chain variable region sequence of theantigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence selected from the group consisting of: SEQ IDNO: 42, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 56, SEQID NO: 60, SEQ ID NO: 64, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76,SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO:96, SEQ ID NO: 100, SEQ ID NO: 104, SEQ ID NO: 108, SEQ ID NO: 112, SEQID NO: 116, SEQ ID NO: 120, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO:132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQID NO: 152, and SEQ ID NO: 156. In another specific embodiment, thelight chain variable region sequence of the antigen binding moieties ofthe immunoconjugate is encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical tosequence selected from the group consisting of: SEQ ID NO: 38, SEQ IDNO: 40, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQID NO: 62, SEQ ID NO: 66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78,SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO:98, SEQ ID NO: 102, SEQ ID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQID NO: 118, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO:134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, andSEQ ID NO: 154. In yet another specific embodiment, the light chainvariable region sequence of the antigen binding moieties of theimmunoconjugate is encoded by a polynucleotide sequence selected fromthe group consisting of: SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,SEQ ID NO: 50, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 62, SEQ ID NO:66, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ IDNO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 102, SEQID NO: 106, SEQ ID NO: 110, SEQ ID NO: 114, SEQ ID NO: 118, SEQ ID NO:122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, and SEQ ID NO: 154.

In another specific embodiment, the immunoconjugate of the inventioncomprises a polypeptide sequence that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected fromthe group of SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO:217, SEQ-ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQID NO: 227, and SEQ ID NO: 229, or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the invention comprises a polypeptide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequenceselected from the group of SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO:235 and SEQ ID NO: 239 or variants thereof that retain functionality. Ina more specific embodiment, the immunoconjugate of the present inventioncomprises a polypeptide sequence that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 211 or SEQ IDNO: 219 or variants thereof that retain functionality, and a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 233 or variants thereof that retainfunctionality. In another specific embodiment, the immunoconjugate ofthe present invention comprises a polypeptide sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to asequence selected from the group of SEQ ID NO: 209, SEQ ID NO: 221, SEQID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227 and SEQ ID NO: 229, orvariants thereof that retain functionality, and a polypeptide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 231 or variants thereof that retainfunctionality. In a further specific embodiment, the immunoconjugate ofthe present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 213 and SEQ ID NO: 235 or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 217 and SEQ ID NO: 239 or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 219 and SEQ ID NO: 233 or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 221 and SEQ ID NO: 231 or variants thereof that retainfunctionality. In-yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 223 and SEQ ID NO: 231 or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 225 and SEQ ID NO: 231 or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 227 and SEQ ID NO: 231 or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 229 and SEQ ID NO: 231 or variants thereof that retainfunctionality. In yet another specific embodiment, the immunoconjugateof the present invention comprises two polypeptide sequences that are atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 211 and SEQ ID NO: 233 or variants thereof that retainfunctionality.

In another specific embodiment, the immunoconjugate of the inventioncomprises a polypeptide sequence that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected fromthe group of SEQ ID NO: 297, SEQ ID NO: 301 and SEQ ID NO: 315, orvariants thereof that retain functionality. In yet another specificembodiment, the immunoconjugate of the invention comprises a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a sequence selected from the group of SEQ ID NO:299, SEQ ID NO: 303 and SEQ ID NO: 317, or variants thereof that retainfunctionality. In a more specific embodiment, the immunoconjugate of thepresent invention comprises a polypeptide sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 297 or a variant thereof that retains functionality, a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 299 or a variant thereof that retainsfunctionality, and a polypeptide sequence that is at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 233 ora variant thereof that retains functionality. In another specificembodiment, the immunoconjugate of the present invention comprises apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 301 or a variant thereofthat retains functionality, a polypeptide sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 303 or a variant thereof that retains functionality, and apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 231 or a variant thereofthat retains functionality. In yet another specific embodiment, theimmunoconjugate of the present invention comprises a polypeptidesequence that is at least about 80W, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 315 or a variant thereof that retainsfunctionality, a polypeptide sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 317 or avariant thereof that retains functionality, and a polypeptide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 233 or a variant thereof that retainsfunctionality.

In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to asequence selected from the group of SEQ ID NO: 210, SEQ ID NO: 212, SEQID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO:224, SEQ ID NO: 226, SEQ ID NO: 228, and SEQ ID NO: 230. In yet anotherspecific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by a polynucleotide sequence selected from the group ofSEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ IDNO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228,and SEQ ID NO: 230. In another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by a polynucleotide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to a sequence selected from the group of SEQ ID NO: 232, SEQID NO: 234, SEQ ID NO: 236, and SEQ ID NO: 240. In yet another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby a polynucleotide sequence selected from the group of SEQ ID NO: 232,SEQ ID NO: 234, SEQ ID NO: 236, and SEQ ID NO: 240.

In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to asequence selected from the group of SEQ ID NO: 298, SEQ ID NO: 302 andSEQ ID NO: 316. In yet another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by a polynucleotide sequenceselected from the group of SEQ ID NO: 298, SEQ ID NO: 302 and SEQ ID NO:316. In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to asequence selected from the group of SEQ ID NO: 300, SEQ ID NO: 304 andSEQ ID NO: 318. In yet another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by a polynucleotide sequenceselected from the group of SEQ ID NO: 300, SEQ ID NO: 304 and SEQ ID NO:318.

In one embodiment, the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for the MelanomaChondroitin Sulfate Proteoglycan (MCSP). In another embodiment, theimmunoconjugate comprises a polypeptide sequence wherein a first Fabheavy chain specific for MCSP shares a carboxy-terminal peptide bondwith a mutant IL-2 polypeptide, which in turn shares a carboxy-terminalpeptide bond with a second Fab heavy chain specific for MCSP. In yetanother embodiment, the immunoconjugate comprises a polypeptide sequencewherein a first Fab light chain specific for MCSP shares acarboxy-terminal peptide bond with an IL-2 molecule, which in turnshares a carboxy-terminal peptide bond with a second Fab light chainspecific for MCSP. In another embodiment, the immunoconjugate comprisesa polypeptide sequence wherein an immunoglobulin heavy chain specificfor MCSP shares a carboxy-terminal peptide bond with a mutant IL-2polypeptide.

In a specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the sequence of either SEQ ID NO: 189 or SEQ ID NO: 193 or variantsthereof that retain functionality. In another specific embodiment, theantigen binding moieties of the immunoconjugate comprise a light chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence of either SEQ ID NO: 191or SEQ ID NO: 197 or variants thereof that retain functionality. In amore specific embodiment, the antigen binding moieties of theimmunoconjugate comprise a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the sequence of either SEQ ID NO: 189 or SEQ ID NO: 193, or variantsthereof that retain functionality, and a light chain variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the sequence of either SEQ ID NO: 191 or SEQ ID NO:197, or variants thereof that retain functionality. In a more specificembodiment, the antigen binding moieties of the immunoconjugate comprisea heavy chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99,% or 100% identical to the sequence of SEQID NO: 189, and a light chain variable region sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thesequence of SEQ ID NO: 191. In another specific embodiment, the antigenbinding moieties of the immunoconjugate comprise a heavy chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequence of SEQ ID NO: 193, and alight chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ IDNO: 191.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 190 orSEQ ID NO: 194. In yet another specific embodiment, the heavy chainvariable region sequence of the antigen binding moieties of theimmunoconjugate is encoded by the polynucleotide sequence of either SEQID NO: 190 or SEQ ID NO: 194. In another specific embodiment, the lightchain variable region sequence of the antigen binding moieties of theimmunoconjugate is encoded by a polynucleotide sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesequence of either SEQ ID NO: 192 or SEQ ID NO: 198. In yet anotherspecific embodiment, the light chain variable region sequence of theantigen binding moieties of the immunoconjugate is encoded by thepolynucleotide sequence of either SEQ ID NO: 192 or SEQ ID NO: 198.

In a specific embodiment, the immunoconjugate of the invention comprisesa polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to either SEQ ID NO: 253 or SEQ ID NO:257, or variants thereof that retain functionality. In another specificembodiment, the immunoconjugate of the invention comprises a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to either SEQ ID NO: 255 or SEQ ID NO: 261, orvariants thereof that retain functionality. In a more specificembodiment, the immunoconjugate of the present invention comprises apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to either SEQ ID NO: 253 or SEQ ID NO:257 or variants thereof that retain functionality, and a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to either SEQ ID NO: 255 or SEQ ID NO: 261, orvariants thereof that retain functionality. In another specificembodiment, the immunoconjugate of the present invention comprises apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 253 or variants thereofthat retain functionality, and a polypeptide sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 255 or variants thereof that retain functionality. In anotherspecific embodiment, the immunoconjugate of the present inventioncomprises a polypeptide sequence that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 257 or variantsthereof that retain functionality, and a polypeptide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toSEQ ID NO: 255 or variants thereof that retain functionality.

In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesequence of either SEQ ID NO: 254 or SEQ ID NO: 258. In yet anotherspecific embodiment, the immunoconjugate comprises a polypeptidesequence encoded by the polynucleotide sequence of either SEQ ID NO: 254or SEQ ID NO: 258. In another specific embodiment, the immunoconjugatecomprises a polypeptide sequence encoded by a polynucleotide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the sequence of either SEQ ID NO: 256 or SEQ ID NO: 262. Inyet another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by the polynucleotide sequence of eitherSEQ ID NO: 256 or SEQ ID NO: 262.

In one embodiment, the immunoconjugate comprises at least one, typicallytwo or more antigen binding moieties that are specific for theCarcinoembryonic Antigen (CEA).

In another embodiment, the immunoconjugate comprises a polypeptidesequence wherein a first Fab heavy chain specific for CEA shares acarboxy-terminal peptide bond with a mutant IL-2 polypeptide, which inturn shares a carboxy-terminal peptide bond with a second Fab heavychain specific for CEA. In yet another embodiment, the immunoconjugatecomprises a polypeptide sequence wherein a first Fab heavy chainspecific for CEA shares a carboxy-terminal peptide bond with a mutantIL-2 polypeptide, which in turn shares a carboxy-terminal peptide bondwith a second Fab heavy chain specific for CEA. In one embodiment, theimmunoconjugate comprises a polypeptide sequence wherein animmunoglobulin heavy chain specific for CEA shares a carboxy-terminalpeptide bond with a mutant IL-2 polypeptide. In a specific embodiment,the antigen binding moieties of the immunoconjugate comprise a heavychain variable region sequence that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:313 or a variant thereof that retains functionality. In another specificembodiment, the antigen binding moieties of the immunoconjugate comprisea light chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ IDNO: 311 or a variant thereof that retains functionality. In a morespecific embodiment, the antigen binding moieties of the immunoconjugatecomprise a heavy chain variable region sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequenceof SEQ ID NO: 313, or a variant thereof that retains functionality, anda light chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ IDNO: 311, or a variant thereof that retains functionality.

In another specific embodiment, the heavy chain variable region sequenceof the antigen binding moieties of the immunoconjugate is encoded by apolynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% identical to the sequence of SEQ ID NO: 314. In yetanother specific embodiment, the heavy chain variable region sequence ofthe antigen binding moieties of the immunoconjugate is encoded by thepolynucleotide sequence of SEQ ID NO: 314. In another specificembodiment, the light chain variable region sequence of the antigenbinding moieties of the immunoconjugate is encoded by a polynucleotidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to the sequence of SEQ ID NO: 312. In yet another specificembodiment, the light chain variable region sequence of the antigenbinding moieties of the immunoconjugate is encoded by the polynucleotidesequence of SEQ ID NO: 312.

In another specific embodiment, the immunoconjugate of the inventioncomprises a polypeptide sequence that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO:319, or variants thereof that retain functionality. In yet anotherspecific embodiment, the immunoconjugate of the invention comprises apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 321, orvariants thereof that retain functionality. In yet another specificembodiment, the immunoconjugate of the invention comprises a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the sequence of SEQ ID NO: 323, or variants thereofthat retain functionality. In a more specific embodiment, theimmunoconjugate of the present invention comprises a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 319 or a variant thereof that retainsfunctionality, a polypeptide sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 321 or avariant thereof that retains functionality, and a polypeptide sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 323 or a variant thereof that retainsfunctionality.

In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 320. In yet another specific embodiment, theimmunoconjugate comprises a polypeptide sequence encoded by thepolynucleotide sequence of SEQ ID NO: 320. In another specificembodiment, the immunoconjugate comprises a polypeptide sequence encodedby a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 322. Inyet another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by the polynucleotide sequence of SEQ IDNO: 322. In another specific embodiment, the immunoconjugate comprises apolypeptide sequence encoded by a polynucleotide sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesequence of SEQ ID NO: 324: In yet another specific embodiment, theimmunoconjugate comprises a polypeptide sequence encoded by thepolynucleotide sequence of SEQ ID NO: 324.

Antigen binding moieties of the invention include those that havesequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to the peptide sequences set forth in SEQ ID NOs23-261 (uneven numbers), 297-303 (uneven numbers), 311 and 313,including functional fragments or variants thereof. The invention alsoencompasses antigen binding moieties comprising sequences of SEQ ID NOs23-261 (uneven numbers), 297-303 (uneven numbers), 311 and 313 withconservative amino acid substitutions.

Polynucleotides

The invention further provides isolated polynucleotides encoding amutant IL-2 polypeptide or an immunoconjugate comprising a mutant IL-2polypeptide as described herein.

Polynucleotides of the invention include those that are at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequences set forth in SEQ ID NOs 2, 4, 5, 6, 8, 9, 10, 12, 13, 14, 16,17, 18, 20, 21, 22, 24-262 (even numbers), 293-296, and 298-324 (evennumbers) including functional fragments or variants thereof.

The polynucleotides encoding mutant IL-2 polypeptides not linked to anon-IL-2 moiety are generally expressed as single polynucleotide thatencodes the entire polypeptide.

In one embodiment, the present invention is directed to an isolatedpolynucleotide encoding a mutant IL-2 polypeptide, wherein thepolynucleotide comprises a sequence that encodes a mutant IL-2 sequenceof SEQ ID NO: 7, 11, 15 or 19. The invention also encompasses anisolated polynucleotide encoding a mutant IL-2 polypeptide, wherein thepolynucleotide comprises a sequence that encodes a mutant IG-2polypeptide of SEQ ID NO: 7, 11, 15 or 19 with conservative amino acidsubstitutions.

In another embodiment, the invention is directed to an isolatedpolynucleotide encoding a mutant IL-2 polypeptide, wherein thepolynucleotide comprises a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequenceselected from the group of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ IDNO: 293, SEQ ID NO: 294, SEQ ID NO: 295 and SEQ ID NO: 296. In anotherembodiment, the invention is directed to an isolated polynucleotideencoding a mutant IL-2 polypeptide, wherein the polynucleotide comprisesa nucleotide sequence selected from the group of SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295 and SEQ IDNO: 296. In another embodiment, the invention is directed to an isolatedpolynucleotide encoding an immunoconjugate or fragment thereof, whereinthe polynucleotide comprises a nucleic acid sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to anucleotide sequence selected from the group of SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295 and SEQ ID NO:296. In another embodiment, the invention is directed to an isolatedpolynucleotide encoding an immunoconjugate or fragment thereof, whereinthe polynucleotide comprises a nucleic acid sequence selected from thegroup of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 293, SEQ ID NO:294, SEQ ID NO: 295 and SEQ ID NO: 296.

The polynucleotides encoding immunoconjugates of the invention may beexpressed as a single polynucleotide that encodes the entireimmunoconjugate or as multiple (e.g., two or more) polynucleotides thatare co-expressed. Polypeptides encoded by polynucleotides that areco-expressed may associate through, e.g., disulfide bonds or other meansto form a functional immunoconjugate. For example, the heavy chainportion of an antigen binding moiety may be encoded by a separatepolynucleotide from the portion of the immunoconjugate comprising thelight chain portion of the antigen binding moiety and the mutant IL-2polypeptide. When co-expressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the antigen bindingmoiety. Alternatively, in another example, the light chain portion ofthe antigen binding moiety could be encoded by a separate polynucleotidefrom the portion of the immunoconjugate comprising the heavy chainportion of the antigen binding moiety and the mutant IL-2 polypeptide.In one embodiment, an isolated polynucleotide of the invention encodes afragment of an immunoconjugate comprising a mutant IL-2 polypeptide andan antigen binding moiety. In one embodiment, an isolated polynucleotideof the invention encodes the heavy chain of an antigen binding moietyand a mutant IL-2 polypeptide. In another embodiment, an isolatedpolynucleotide of the invention encodes the light chain of an antigenbinding moiety and a mutant IL-2 polypeptide.

In a specific embodiment, an isolated polynucleotide of the inventionencodes a fragment of an immunoconjugate comprising at least one mutantIL-2 polypeptide, and at least one, preferably two or more antigenbinding moieties, wherein a first mutant IL-2 polypeptide shares anamino- or carboxy-terminal peptide bond with a first antigen bindingmoiety and a second antigen binding moiety shares an amino- orcarboxy-terminal peptide bond with either the first mutant IL-2polypeptide or the first antigen binding moiety. In a one embodiment,the antigen binding moieties are independently selected from the groupconsisting of a Fv molecule, particularly a scFv molecule, and a Fabmolecule. In another specific embodiment, the polynucleotide encodes theheavy chains of two of the antigen binding moieties and one mutant IL-2polypeptide. In another specific embodiment, the polynucleotide encodesthe light chains of two of the antigen binding moieties and one mutantIL-2 polypeptide. In another specific embodiment, the polynucleotideencodes one light chain of one of the antigen binding moieties, oneheavy chain of a second antigen binding moiety and one mutant IL-2polypeptide.

In another specific embodiment, an isolated polynucleotide of theinvention encodes a fragment of an immunoconjugate, wherein thepolynucleotide encodes the heavy chains of two Fab molecules and amutant IL-2 polypeptide. In another specific embodiment, an isolatedpolynucleotide of the invention encodes a fragment of animmunoconjugate, wherein the polynucleotide encodes the light chains oftwo Fab molecules and a mutant IL-2 polypeptide. In another specificembodiment an isolated polynucleotide of the invention encodes afragment of an immunoconjugate, wherein the polynucleotide encodes theheavy chain of one Fab molecule, the light chain of second Fab moleculeand a mutant IL-2 polypeptide.

In one embodiment, an isolated polynucleotide of the invention encodesan immunoconjugate comprising at least one mutant IL-2 polypeptide,joined at its amino- and carboxy-terminal amino acids to one or morescFv molecules.

In one embodiment, an isolated polynucleotide of the invention encodes afragment of an immunoconjugate, wherein the polynucleotide encodes theheavy chain of an immunoglobulin molecule, particularly an IgG molecule,more particularly an IgG, molecule, and a mutant IL-2 polypeptide. In amore specific embodiment, the isolated polynucleotide encodes a theheavy chain of an immunoglobulin molecule and a mutant IL-2 polypeptide,wherein the mutant IL-2 polypeptide shares a amino-terminal peptide bondwith the immunoglobulin heavy chain.

In another embodiment, the present invention is directed to an isolatedpolynucleotide encoding an immunoconjugate or fragment thereof, whereinthe polynucleotide comprises a sequence that encodes a variable regionsequence as shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 231, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,195, 197, 311 or 313. In another embodiment, the present invention isdirected to an isolated polynucleotide encoding an immunoconjugate orfragment thereof, wherein the polynucleotide comprises a sequence thatencodes a polypeptide sequence as shown in SEQ ID NO: 199, 201, 203,205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231,233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259,261, 297, 299, 301, 303, 315, 317, 319, 321 or 323. In anotherembodiment, the invention is further directed to an isolatedpolynucleotide encoding an immunoconjugate or fragment thereof, whereinthe polynucleotide comprises a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence shownin SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256,258, 260, 262, 298, 300, 302, 304, 312, 314, 316, 318, 320, 322 or 324.In another embodiment, the invention is directed to an isolatedpolynucleotide encoding an immunoconjugate or fragment thereof, whereinthe polynucleotide comprises a nucleic acid sequence shown in SEQ ID NO:24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 298,300, 302, 304, 312, 314, 316, 318, 320, 322 or 324. In anotherembodiment, the invention is directed to an isolated polynucleotideencoding an immunoconjugate or fragment thereof, wherein thepolynucleotide comprises a sequence that encodes a variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical to an amino acid sequence of SEQ ID NO: 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,231, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 311 or 313. In another embodiment, theinvention is directed to an isolated polynucleotide encoding animmunoconjugate or fragment thereof, wherein the polynucleotidecomprises a sequence that encodes a polypeptide sequence that is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an aminoacid sequence of SEQ ID NO: 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 297, 299, 301, 303, 315,317, 319, 321 or 323. The invention encompasses an isolatedpolynucleotide encoding an immunoconjugate or fragment thereof, whereinthe polynucleotide comprises a sequence that encodes the variable regionsequences of SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 231, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,311 or 313 with conservative amino acid substitutions. The inventionalso encompasses an isolated polynucleotide encoding an immunoconjugateof the invention or fragment thereof, wherein the polynucleotidecomprises a sequence that encodes the polypeptide sequences of SEQ IDNO: 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, −0.255, 257, 259, 261, 297, 299, 301, 303, 315, 317; 319, 321 or323 with conservative amino acid substitutions.

In certain embodiments the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention is RNA, forexample, in the form of messenger RNA (mRNA). RNA of the presentinvention may be single stranded or double stranded.

Recombinant Methods

Mutant-IL-2 polypeptides of the invention can be prepared by deletion,substitution, insertion or modification using genetic or chemicalmethods well known in the art. Genetic methods may include site-specificmutagenesis of the encoding DNA sequence, PCR, gene synthesis, and thelike. The correct nucleotide changes can be verified for example bysequencing. In this regard, the nucleotide sequence of native IL-2 hasbeen described by Taniguchi et al. (Nature 302, 305-(1983)) and nucleicacid encoding human IL-2 is available from public depositories such asthe American Type Culture Collection (Rockville Md.). The sequence ofnative human IL-2 is shown in SEQ ID NO: 1. Substitution or insertionmay involve natural as well as non-natural amino acid residues. Aminoacid modification includes well known methods of chemical modificationsuch as the addition of glycosylation sites or carbohydrate attachments,and the like.

Mutant IL-2 polypeptides and immunoconjugates of the invention may beobtained, for example, by solid-state peptide synthesis or recombinantproduction. For recombinant production one or more polynucleotideencoding said mutant IL-2 polypeptide or immunoconjugate (fragment),e.g., as described above, is isolated and inserted into one or morevectors for further cloning and/or expression in a host cell. Suchpolynucleotide may be readily isolated and sequenced using conventionalprocedures. In one embodiment a vector, preferably an expression vector,comprising one or more of the polynucleotides of the invention isprovided. Methods which are well known to those skilled in the art canbe used to construct expression vectors containing the coding sequenceof a mutant IL-2 polypeptide or immunoconjugate (fragment) along withappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y (1989). The expression vector can be part of aplasmid, virus, or may be a nucleic acid fragment. The expression vectorincludes an expression cassette into which the polynucleotide encodingthe IL-2 mutant or the immunoconjugate (fragment) (i.e. the codingregion) is cloned in operable association with a promoter and/or othertranscription or translation control elements. As used herein, a “codingregion” is a portion of nucleic acid which consists of codons translatedinto amino acids. Although a “stop codon” (TAG, TGA, or TAA) is nottranslated into an amino acid, it may be considered to be part of acoding region, if present, but any flanking sequences, for examplepromoters, ribosome binding sites, transcriptional terminators, introns,5′ and 3′ untranslated regions, and the like, are not part of a codingregion. Two or more coding regions can be present in a singlepolynucleotide construct, e.g. on a single vector, or in separatepolynucleotide constructs, e.g. on separate (different) vectors.Furthermore, any vector may contain a single coding region, or maycomprise two or more coding regions, e.g. a vector of the presentinvention may encode one or more polyproteins, which are post- orco-translationally separated into the final proteins via proteolyticcleavage. In addition, a vector, polynucleotide, or nucleic acid of theinvention may encode heterologous coding regions, either fused ofunfused to a first or second polynucleotide encoding the polypeptides ofthe invention, or variant or derivative thereof. Heterologous codingregions include without limitation specialized elements or motifs, suchas a secretory signal peptide or a heterologous functional domain. Anoperable association is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences in sucha way as to place expression of the gene product under the influence orcontrol of the regulatory sequence(s). Two DNA fragments (such as apolypeptide coding region and a promoter associated therewith) are“operably associated” if induction of promoter function results in thetranscription of mRNA encoding the desired gene product and if thenature of the linkage between the two DNA fragments does not interferewith the ability of the expression regulatory sequences to direct theexpression of the gene product or interfere with the ability of the DNAtemplate to be transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if the promoterwas capable of effecting transcription of that nucleic acid. Thepromoter may be a cell-specific promoter that directs substantialtranscription of the DNA only in predetermined cells. Othertranscription control elements, besides a promoter, for exampleenhancers, operators, repressors, and transcription termination signals,can be operably associated with the polynucleotide to directcell-specific transcription. Suitable promoters and other transcriptioncontrol regions are disclosed herein. A variety of transcription controlregions are known to those skilled in the art. These include, withoutlimitation, transcription control regions, which function in vertebratecells, such as, but not limited to, promoter and enhancer segments fromcytomegaloviruses (e.g. the immediate early promoter, in conjunctionwith intron-A), simian virus 40 (e.g. the early promoter), andretroviruses (such as, e.g. Rous sarcoma virus). Other transcriptioncontrol regions include those derived from vertebrate genes such asactin, heat shock protein, bovine growth hormone and rabbit β-globin, aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control regionsinclude tissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, a varietyof translation control elements are known to those of ordinary skill inthe art. These include, but are not limited to ribosome binding sites,translation initiation and termination codons, and elements derived fromviral systems (particularly an internal ribosome entry site, or IRES,also referred to as a CITE sequence). The expression cassette may alsoinclude other features such as an origin of replication, and/orchromosome integration elements such as retroviral long terminal repeats(LTRs), or adeno-associated viral (AAV) inverted terminal repeats(ITRs).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. For example, if secretionof the mutant IL-2 polypeptide is desired, DNA encoding a signalsequence may be placed upstream of the nucleic acid encoding the matureamino acids of the mutant IL-2. The same applies to immunoconjugates ofthe invention or fragments thereof. According to the signal hypothesis,proteins secreted by mammalian cells have a signal peptide or secretoryleader sequence which is cleaved from the mature protein once export ofthe growing protein chain across the rough endoplasmic reticulum hasbeen initiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which is cleavedfrom the translated polypeptide to produce a secreted or “mature” formof the polypeptide. For example, human IL-2 is translated with a 20amino acid signal sequence at the N-terminus of the polypeptide, whichis subsequently cleaved off to produce the mature, 133 amino acid humanIL-2. In certain embodiments, the native signal peptide, e.g. the IL-2signal peptide or an immunoglobulin heavy chain or light chain signalpeptide is used, or a functional derivative of that sequence thatretains the ability to direct the secretion of the polypeptide that isoperably associated with it. Alternatively, a heterologous mammaliansignal peptide, or a functional derivative thereof, may be used. Forexample, the wild-type leader sequence may be substituted with theleader sequence of human tissue plasminogen activator (TPA) or mouseβ-glucuronidase. Exemplary amino acid and polynucleotide sequences ofsecretory signal peptides are shown in SEQ ID NOs 236-273.

DNA encoding a short protein sequence that could be used to facilitatelater purification (e.g. a histidine tag) or assist in labeling the IL-2mutant or immunoconjugate may be included within or at the ends of theIL-2 mutant or immunoconjugate (fragment) encoding polynucleotide.

In a further embodiment, a host cell comprising one or morepolynucleotides of the invention is provided. In certain embodiments ahost cell comprising one or more vectors of the invention is provided.The polynucleotides and vectors may incorporate any of the features,singly or in combination, described herein in relation topolynucleotides and vectors, respectively. In one such embodiment a hostcell comprises (e.g. has been transformed or transfected with) a vectorcomprising a polynucleotide that encodes an amino acid sequencecomprising the mutant IL-2 polypeptide of the invention. As used herein,the term “host cell” refers to any kind of cellular system which can beengineered to generate the mutant IL-2 polypeptides or immunoconjugatesof the invention or fragments thereof. Host cells suitable forreplicating and for supporting expression of mutant IL-2 polypeptides orimmunoconjugates are well known in the art. Such cells may betransfected or transduced as appropriate with the particular expressionvector and large quantities of vector containing cells can be grown forseeding large scale fermenters to obtain sufficient quantities of theIL-2 mutant or immunoconjugate for clinical applications. Suitable hostcells include prokaryotic microorganisms, such as E. coli, or variouseukaryotic cells, such as Chinese hamster ovary cells (CHO), insectcells, or the like. For example, polypeptides may be produced inbacteria in particular when glycosylation is not needed. Afterexpression, the polypeptide may be isolated from the bacterial cellpaste in a soluble fraction and can be further purified. In addition toprokaryotes, eukaryotic microbes such as filamentous fungi or yeast aresuitable cloning or expression hosts for polypeptide-encoding vectors,including fungi and yeast strains whose glycosylation pathways have been“humanized,” resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gemgross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215(2006). Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cells includeplant and insect cells. Numerous baculoviral strains have beenidentified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plant cellcultures can also be utilized as hosts. See e.g. U.S. Pat. Nos.5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describingPLANTIBODIES™ technology for producing antibodies in transgenic plants).Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J Gen Virol 36, 59 (1977)),baby hamster kidney cells (BHK), mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol Reprod 23, 243-251 (1980)), monkeykidney cells (CVI), African green monkey kidney cells (VERO-76), humancervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor cells (MMT 060562), TRI cells (as described,e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5cells, and FS4 cells. Other useful mammalian host cell lines includeChinese hamster ovary (CHO) cells, including dhfr CHO cells (Urlaub etal., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell linessuch as YO, NS0, P3X63 and Sp2/0. For a review of certain mammalian hostcell lines suitable for protein production, see, e.g., Yazaki and Wu,Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,Totowa, N.J.), pp. 255-268 (2003). Host cells include cultured cells,e.g., mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant or animaltissue. In one embodiment, the host cell is a eukaryotic cell,preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell,a human embryonic kidney (HEK) cell or lymphoid cell (e.g., YO, NS0,Sp20 cell).

Standard technologies are known in the art to express foreign genes inthese systems. Cells expressing a mutant-IL-2 polypeptide fused toeither the heavy or the light chain of an antigen binding domain such asan antibody, may be engineered so as to also express the other of theantibody chains such that the expressed mutant IL-2 fusion product is anantibody that has both a heavy and a light chain.

In one embodiment, a method of producing a mutant IL-2 polypeptide or animmunoconjugate according to the invention is provided, wherein themethod comprises culturing a host cell comprising a polynucleotideencoding the mutant IL-2 polypeptide or immunoconjugate, as providedherein, under conditions suitable for expression of the mutant IL-2polypeptide or immunoconjugate, and optionally recovering the mutantIL-2 polypeptide or immunoconjugate from the host cell (or host cellculture medium).

In certain embodiments according to the invention the mutant IL-2polypeptide is linked to at least one non-IL-2 moiety. An IL-2 mutantcan be prepared where the mutant IL-2 polypeptide segment is linked toone or more molecules such as a polypeptide, protein, carbohydrate,lipid, nucleic acid, polynucleotide or molecules that are combinationsof these molecules (e.g. glycoproteins, glycolipids etc.). The mutantIL-2 polypeptide also may be linked to an organic moiety, inorganicmoiety or pharmaceutical drug. As used herein, a pharmaceutical drug isan organic containing compound of about 5,000 daltons or less. Themutant IL-2 polypeptide also may be linked to any biological agentincluding therapeutic compounds such as anti-neoplastic agents,anti-microbial agents, hormones, immunomodulators, anti-inflammatoryagents and the like. Also included are radioisotopes such as thoseuseful for imaging as well as for therapy.

The mutant IG-2 polypeptide may also be linked to multiple molecules ofthe same type or to more than one type of molecule. In certainembodiments, the molecule that is linked to IL-2 can confer the abilityto target the IL-2 to specific tissues or cells in an animal, and isreferred to herein as a “targeting moiety”. In these embodiments, thetargeting moiety may have affinity for a ligand or receptor in thetarget tissue or cell, thereby directing the IL-2 to the target tissueor cell. In a particular embodiment the targeting moiety directs theIL-2 to a tumor. Targeting moieties include, for example, antigenbinding moieties (e.g. antibodies and fragments thereof) specific forcell surface or intracellular proteins, ligands of biological receptors,and the like. Such antigen binding moieties may be specific for tumorassociated antigens such as the ones described herein.

A mutant IG-2 polypeptide may be genetically fused to anotherpolypeptide, e.g. a single chain antibody, or (part of) an antibodyheavy or light chains, or may be chemically conjugated to anothermolecule. Fusion of a mutant IL-2 polypeptide to part of an antibodyheavy chain is described in the Examples. An IL-2 mutant which is afusion between a mutant IG-2 polypeptide and another polypeptide can bedesigned such that the IL-2 sequence is fused directly to thepolypeptide or indirectly through a linker sequence. The composition andlength of the linker may be determined in accordance with methods wellknown in the art and may be tested for efficacy. An example of a linkersequence between IL-2 and an antibody heavy chain is found in thesequences shown e.g. in SEQ ID NOs 209, 211, 213 etc. Additionalsequences may also be included to incorporate a cleavage site toseparate the individual components of the fusion if desired, for examplean endopeptidase recognition sequence. In addition, an IL-2 mutant orfusion protein thereof may also be synthesized chemically using methodsof polypeptide synthesis as is well known in the art (e.g. Merrifieldsolid phase synthesis). Mutant IL-2 polypeptides may be chemicallyconjugated to other molecules, e.g. another polypeptide, using wellknown chemical conjugation methods. Bi-functional cross-linking reagentssuch as homofunctional and heterofunctional cross-linking reagents wellknown in the art can be used for this purpose. The type of cross-linkingreagent to use depends on the nature of the molecule to be coupled toIL-2 and can readily be identified by those skilled in the art.Alternatively, or in addition, mutant IL-2 and/or the molecule to whichit is intended to be conjugated may be chemically derivatized such thatthe two can be conjugated in a separate reaction as is also well knownin the art.

In certain embodiments the mutant IL-2 polypeptide is linked to one ormore antigen binding moieties (i.e. is part of an immunoconjugate)comprising at least an antibody variable region capable of binding anantigenic determinant. Variable regions can form part of and be derivedfrom naturally or non-naturally occurring antibodies and fragmentsthereof. Methods to produce polyclonal antibodies and monoclonalantibodies are well known in the art (see e.g. Harlow and Lane,“Antibodies, a laboratory manual”, Cold Spring Harbor Laboratory, 1988).Non-naturally occurring antibodies can be constructed using solidphase-peptide synthesis, can be produced recombinantly (e.g. asdescribed in U.S. Pat. No. 4,186,567) or can be obtained, for example,by screening combinatorial libraries comprising variable heavy chainsand variable light chains (see e.g. U.S. Pat. No. 5,969,108 toMcCafferty). Immunoconjugates, antigen binding moieties and methods forproducing the same are also described in detail in PCT publication no.WO 2011/020783, the entire content of which is incorporated herein byreference.

Any animal species of antibody, antibody fragment, antigen bindingdomain or variable region can be linked to a mutant IL-2 polypeptide.Non-limiting antibodies, antibody fragments, antigen binding-domains orvariable regions useful in the present invention can be of murine,primate, of human origin. If the mutant IL-2/antibody conjugate orfusion is intended for human use, a chimeric form of the antibody may beused wherein the constant regions of the antibody are from a human. Ahumanized or fully human form of the antibody can also be prepared inaccordance with methods well known in the art (see e. g. U.S. Pat. No.5,565,332 to Winter). Humanization may be achieved by various methodsincluding, but not limited to (a) grafting the non-human (e.g., donorantibody) CDRs onto human (e.g. recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g. those that are important for retaining good antigenbinding affinity or antibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or α-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (c) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332, 323-329(1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81,6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988);Verhoeyen at al., Science 239, 1534-1536 (1988); Padlan, Molec Immun31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36, 43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36,61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000)(describing the “guided selection” approach to FR shuffling). Humanantibodies and human variable regions can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) andLonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regionscan form part of and be derived from human monoclonal antibodies made bythe hybridoma method (see e.g. Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Human antibodies and human variable regions may also be prepared byadministering an immunogen to a transgenic animal that has been modifiedto produce intact human antibodies or intact antibodies with humanvariable regions in response to antigenic challenge (see e.g. Lonberg,Nat Biotech 23, 1117-1125 (2005). Human antibodies and human variableregions may also be generated by isolating Fv clone variable regionsequences selected from human-derived phage display libraries (see e.g.,Hoogenboom et al. in Methods in Molecular Biology 178, 1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001); and McCafferty et al.,Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv (scFv)fragments or as Fab fragments. A detailed description of the preparationof antigen binding moieties for immunoconjugates by phage display can befound in the Examples appended to PCT publication no. WO 2011/020783.

In certain embodiments, the antigen binding moieties useful in thepresent invention are engineered to have enhanced binding affinityaccording to, for example, the methods disclosed in PCT publication-no.WO 2011/020783 (see Examples relating to affinity maturation) or U.S.Pat. Appl. Publ. No. 2004/0132066, the entire contents of which arehereby incorporated by reference. The ability of the immunoconjugate ofthe invention to bind to a specific antigenic determinant can bemeasured either through an enzyme-linked immunosorbent assay (ELISA) orother techniques familiar to one of skill in the art, e.g. surfaceplasmon resonance technique (analyzed on a BIACORE T100 system)(Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditional bindingassays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays maybe used to identify an antibody, antibody fragment, antigen bindingdomain or variable domain that competes with a reference antibody forbinding to a particular antigen, e.g. an antibody that competes with theL19 antibody for binding to the Extra Domain B of fibronectin (EDB). Incertain embodiments, such a competing antibody binds to the same epitope(e.g. a linear or a conformational epitope) that is bound by thereference antibody. Detailed exemplary methods for mapping an epitope towhich an antibody binds are provided in Morris (1996) “Epitope MappingProtocols,” in Methods in Molecular Biology vol. 66 (Humana Press,Totowa, N.J.). In an exemplary competition assay, immobilized antigen(e.g. EDB) is incubated in a solution comprising a first labeledantibody that binds to the antigen (e.g. L19 antibody) and a secondunlabeled antibody that is being tested for its ability to compete withthe first antibody for binding to the antigen. The second antibody maybe present in a hybridoma supernatant. As a control, immobilized antigenis incubated in a solution comprising the first labeled antibody but notthe second unlabeled antibody. After incubation under conditionspermissive for binding of the first antibody to the antigen, excessunbound antibody is removed, and the amount of label associated withimmobilized antigen is measured. If the amount of label associated withimmobilized antigen is substantially reduced in the test sample relativeto the control sample, then that indicates that the second antibody iscompeting with the first antibody for binding to the antigen. See Harlowand Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.).

Further chemical modification of the mutant IL-2 mutant orimmunoconjugate of the invention may be desirable. For example, problemsof immunogenicity and short half-life may be improved by conjugation tosubstantially straight chain polymers such as polyethylene glycol (PEG)or polypropylene glycol (PPG) (see e.g. WO 87/00056).

IL-2 mutants and immunoconjugates prepared as described herein may bepurified by art-known techniques such as high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography, size exclusion chromatography, and the like.The actual conditions used to purify a particular protein will depend,in part, on factors such as net charge, hydrophobicity, hydrophilicityetc., and will be apparent to those having skill in the art. Foraffinity chromatography purification an antibody, ligand, receptor orantigen can be used to which the mutant IL-2 polypeptide orimmunoconjugate binds. For example, an antibody which specifically bindsthe mutant IL-2 polypeptide may be used. For affinity chromatographypurification of immunoconjugates of the invention, a matrix with proteinA or protein G may be used. For example, sequential Protein A or Gaffinity chromatography and size exclusion chromatography can be used toisolate an immunoconjugate essentially as described in the Examples. Thepurity of the mutant IL-2 polypeptides and fusion proteins thereof canbe determined by any of a variety of well known analytical methodsincluding gel electrophoresis, high pressure liquid chromatography, andthe like. For example, the heavy chain fusion proteins expressed asdescribed in the Examples were shown to be intact and properly assembledas demonstrated by reducing SDS-PAGE (see e.g. FIG. 14). Two bands wereresolved at approximately Mr 25,000 and Mr 60,000, corresponding to thepredicted molecular weights of the immunoglobulin light chain and heavychain/IL-2 fusion protein.

Assays

Mutant IL-2 polypeptides and immunoconjugates provided herein may beidentified, screened for, or characterized for their physical/chemicalproperties and/or biological activities by various assays known in theart.

Affinity Assays

The affinity of the mutant or wild-type IL-2 polypeptide for variousforms of the IL-2 receptor can be determined in accordance with themethod set forth in the Examples by surface plasmon resonance (SPR),using standard instrumentation such as a BIAcore instrument (GEHealthcare), and receptor subunits such as may be obtained byrecombinant expression (see e.g. Shanafelt et al., Nature Biotechnol 18,1197-1202 (2000)). A recombinant IL-2 receptor β/γ-subunit heterodimercan be generated by fusing each of the subunits to an antibody Fc domainmonomer modified by the knobs-into-holes technology (see e.g. U.S. Pat.No. 5,731,168) to promote heterodimerization of the appropriate receptorsubunit/Fc fusion proteins (see SEQ ID NOs 102 and 103). Alternatively,binding affinity of IL-2 mutants for different forms of the IL-2receptor may be evaluated using cell lines known to express one or theother such form of the receptor. A specific illustrative and exemplaryembodiment for measuring binding affinity is described in the followingand in the Examples below. According to one embodiment, K_(D) ismeasured by surface plasmon resonance using a BIACORE® T100 machine (GEHealthcare) at 25° C. with IL-2 receptors immobilized on CM5 chips.Briefly, carboxymethylated dextran biosensor chips (CM5, GE Healthcare)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Recombinant IL-2 receptor is diluted with 10 mMsodium acetate, pH 5.5, to 0.5-30 μg/ml before injection at a flow rateof 10 μl/minute to achieve approximately 200-1000 (for IL-2R α-subunit)or 500-3000 (for IL-2R βγ knobs-into-holes heterodimer) response units(RU) of coupled protein. Following the injection of IL-2 receptor, 1 Methanolamine is injected to block unreacted groups. For kineticsmeasurements, three-fold serial dilutions of mutant IL-2 polypeptide orimmunoconjugate (range between ˜0.3 nM to 300 nM) are injected inHBS-EP+(GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05%Surfactant P20, pH 7.4) at 25° C. at a flow rate of approximately 30μl/min. Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIACORE®T100 Evaluation Software version 1.1.1) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (K_(D)) is calculated as the ratio K_(off)/k_(on). See, e.g.,Chen et al., J Mol Biol 293, 865-881 (1999).

Binding of immunoconjugates of the invention to Fc receptors can beeasily determined e.g. by ELISA, or by Surface Plasmon Resonance (SPR)using standard instrumentation such as a BIAcore instrument (GEHealthcare), and Fc receptors such as may be obtained by recombinantexpression. Alternatively, binding affinity of Fc domains orimmunoconjugates comprising an Fc domain for Fc receptors may beevaluated using cell lines known to express particular Fc receptors,such as NK cells expressing FcγIIIa receptor. According to oneembodiment, K_(D) is measured by surface plasmon resonance using aBIACORE® T100 machine (GE Healthcare) at 25° C. with Fc receptorsimmobilized on CM5 chips. Briefly, carboxymethylated dextran biosensbrchips (CM5, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Recombinant Fc receptor is diluted with 10 mM sodium acetate, pH 5.5, to0.5-30 μg/ml before injection at a flow rate of 10 μl/minute to achieveapproximately 100-5000 response units (RU) of coupled protein. Followingthe injection of the Fc receptor, 1 M ethanolamine is injected to blockunreacted groups. For kinetics measurements, three- to five-fold serialdilutions of immunoconjugate (range between ˜0.01 nM to 300 nM) areinjected in HBS-EP+(GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA,0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of approximately30-50 μl/min. Association rates (k_(on)) and dissociation rates(k_(off)) are calculated using a simple one-to-one Langmuir bindingmodel (BIACORE® T100 Evaluation Software version 1.1.1) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

Activity Assays

The ability of an IL-2 mutant to bind to IL-2 receptors may beindirectly measured by assaying the effects of immune activation thatoccur downstream of receptor binding.

In one aspect, assays are provided for identifying mutant IL-2polypeptides having biological activity. Biological activities mayinclude, e.g., the ability to induce proliferation of IL-2receptor-bearing T and/or NK cells, the ability to induce IL-2 signalingin IL-2 receptor-bearing T and/or NK cells, the ability to generateinterferon (IFN)-γ as a secondary cytokine by NK cells, a reducedability to induce elaboration of secondary cytokines, particularly IL-10and TNF-α, by peripheral blood mononuclear cells (PBMCs), a reducedability to induce apoptosis in T cells, the ability to induce tumorregression and/or improve survival, and a reduced toxicity profile,particularly reduced vasopermeability, in vivo. Mutant IL-2 polypeptideshaving such biological activity in vivo and/or in vitro are alsoprovided.

In certain embodiments, a mutant IL-2 polypeptide of the invention istested for such biological activity. A variety of methods are well knownthe art for determining biological activities of IL-2, and also detailsfor many of these methods are disclosed in the Examples appendedherewith. The Examples provide a suitable assay for testing IL-2 mutantsof the invention for their ability to generate IFN-γ by NK cells.Cultured NK cells are incubated with the mutant IL-2 polypeptide orimmunoconjugates of the invention, and IFN-γ concentration in theculture medium is subsequently measured by ELISA.

IL-2 induced signaling induces several signaling pathways, and involvesJAK (Janus kinase) and STAT (signal transducer and activator oftranscription) signaling molecules. The interaction of IL-2 with thereceptor β- and γ-subunits leads to phosphorylation of the receptor andof JAK1 and JAK3, which are associated with the β- and γ-subunit,respectively. STAT5 then associates with the phosphorylated receptor andis phosphorylated itself on a crucial tyrosin residue. This results inthe dissociation of STAT5 from the receptor, dimerization of STAT5 andtranslocation of the STAT5 dimers to the nucleus where they promote thetranscription of target genes. The ability of mutant IL-2 polypeptidesto induce signaling through the IL-2 receptor can thus be assessed, forexample, by measuring phosphorylation of STAT5. Details of this methodare disclosed in the Examples. PBMCs are treated with mutant IL-2polypeptides or immunoconjugates of the invention and levels ofphosphorylated STAT5 are determined by flow cytometry.

Proliferation of T cells or NK cells in response to IL-2 may be measuredby incubating T cells or NK cells isolated from blood with mutant IL-2polypeptides or immunoconjugates of the invention, followed bydetermination of the ATP content in lysates of the treated cells. Beforetreatment, T cells may be pre-stimulated with phytohemagglutinin(PHA-M). This assay, described in the Examples, allows sensitivequantitation of the number of viable cells, however there are numeroussuitable alternative assays known in the art (e.g. [³H]-thymidineincorporation assay, Cell Titer Glo ATP assays, Alamar Blue assay, WST-1assay, MTT assay).

An assay for determination of apoptosis of T cells and AICD is alsoprovided in the Examples, wherein T cells are treated with anapoptosis-inducing antibody after the incubation with the mutant IL-2polypeptides or immunoconjugates of the invention and apoptotic cellsare quantified by flow cytometric detection of phosphatidylserine/annexin exposure. Other assays are known in the art.

The effects of mutant IL-2 on tumor growth and survival can be assessedin a variety of animal tumor models known in the art. For example,xenografts of human cancer cell lines can be implanted toimmunodeficient mice, and treated with mutant IL-2 polypeptides orimmunoconjugates of the invention, as described in the Examples.

Toxicity of mutant IL-2 polypeptides and immunoconjugates of theinvention in vivo can be determined based on mortality, in-lifeobservations (visible symptoms of adverse effects, e.g. behaviour, bodyweight, body temperature) and clinical and anatomical pathology (e.g.measurements of blood chemistry values and/or histopathologicalanalyses). Vasopermeability induced by treatment with IL-2 can beexamined in a pretreatment vasopermeability animal model. In general,the IL-2 mutant or immunoconjugate of the invention is administered to asuitable animal, e.g. a mouse, and at a later time the animal isinjected with a vascular leak reporter molecule whose dissemination fromthe vasculature reflects the extent of vascular permeability. Thevascular leak reporter molecule is preferably large enough to revealpermeability with the wild-type form of IL-2 used for pretreatment. Anexample of a vascular leak reporter molecule can be a serum protein suchas albumin or an immunoglobulin. The vascular leak reporter moleculepreferably is detectably labeled such as with a radioisotope tofacilitate quantitative determination of the molecule's tissuedistribution. Vascular permeability may be measured for vessels presentin any of a variety of internal body organs such as liver, lung, and thelike, as well as a tumor, including a tumor that is xenografted. Lung isa preferred organ for measuring vasopermeability of full-length IL-2mutants.

Compositions, Formulations, and Routes of Administration

In a further aspect, the invention provides pharmaceutical compositionscomprising any of the mutant IL-2 polypeptides or immunoconjugatesprovided herein, e.g., for use in any of the below therapeutic methods.In one embodiment, a pharmaceutical composition comprises any of themutant IL-2 polypeptides or immunoconjugates provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition comprises any of the mutant IL-2 polypeptidesor immunoconjugates provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Further provided is a method of producing a mutant IL-2 polypeptide oran immunoconjugate of the invention in a form suitable foradministration in vivo, the method comprising (a) obtaining a mutantIL-2 polypeptide or immunoconjugate according to the invention, and (b)formulating the mutant IL-2 polypeptide or immunoconjugate with at leastone pharmaceutically acceptable carrier, whereby a preparation of mutantIL-2 polypeptide or immunoconjugate is formulated for administration invivo.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more mutant IL-2 polypeptideor immunoconjugate dissolved or dispersed in a pharmaceuticallyacceptable carrier. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that aregenerally non-toxic to recipients at the dosages and concentrationsemployed, i.e. do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one mutant IL-2 polypeptide or immunoconjugate andoptionally an additional active ingredient will be known to those ofskill in the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards or correspondingauthorities in other countries. Preferred compositions are lyophilizedformulations or aqueous solutions. Exemplary IL-2 compositions aredescribed in U.S. Pat. Nos. 4,604,377 and 4,766,106. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,buffers, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g. antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, antioxidants,proteins, drugs, drug stabilizers, polymers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp.1289-1329, incorporated herein by reference). Except insofar as anyconventional carrier is incompatible with the active ingredient, its usein the therapeutic or pharmaceutical compositions is contemplated.

The composition may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. Mutant IL-2 polypeptides or immunoconjugates of the presentinvention (and any additional therapeutic agent) can be administeredintravenously, intradermally, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrasplenically, intrarenally, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctivally, intravesicularily, mucosally, intrapericardially,intraumbilically, intraocularally, orally, topically, locally, byinhalation (e.g. aerosol inhalation), injection, infusion, continuousinfusion, localized perfusion bathing target cells directly, via acatheter, via a lavage, in cremes, in lipid compositions (e.g.liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference). Parenteral administration, inparticular intravenous injection, is most commonly used foradministering polypeptide molecules such as the mutant IL-2 polypeptidesand immunoconjugates of the invention.

Parenteral compositions include those designed for administration byinjection, e.g. subcutaneous, intradermal, intralesional, intravenous,intraarterial intramuscular, intrathecal or intraperitoneal injection.For injection, the mutant IL-2 polypeptides and immunoconjugates of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringerssolution, or physiological saline buffer. The solution may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the mutant IL-2 polypeptides and immunoconjugatesmay be in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. Sterile injectable solutions areprepared by incorporating the IL-2 polypeptides or immunoconjugates ofthe invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated below, as required.Sterility may be readily accomplished, e.g., by filtration throughsterile filtration membranes. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The composition must be stable under theconditions of manufacture and storage, and preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Itwill be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Suitable pharmaceutically acceptable carriers include, but are notlimited to: buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Aqueous injectionsuspensions may contain compounds which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, dextran,or the like. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl cleats or triglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(18th Ed. Mack Printing Company, 1990). Sustained-release preparationsmay be prepared. Suitable examples of sustained-release preparationsinclude semipermeable matrices of solid hydrophobic polymers containingthe polypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. In particular embodiments, prolonged absorptionof an injectable composition can be brought about by the use in thecompositions of agents delaying absorption, such as, for example,aluminum monostearate, gelatin or combinations thereof.

In addition to the compositions described previously, theimmunoconjugates may also be formulated as a depot preparation. Suchlong acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection.

Thus, for example, the mutant IL-2 polypeptides and immunoconjugates maybe formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

Pharmaceutical compositions comprising the mutant IL-2 polypeptides andimmunoconjugates of the invention may be manufactured by means ofconventional mixing, dissolving emulsifying, encapsulating, entrappingor lyophilizing processes. Pharmaceutical compositions may be formulatedin conventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The mutant IL-2 polypeptides and immunoconjugates may be formulated intoa composition in a free acid or base, neutral or salt form.Pharmaceutically acceptable salts are salts that substantially retainthe biological activity of the free acid or base. These include the acidaddition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Pharmaceutical salts tend to be more soluble in aqueous andother protic solvents than are the corresponding free base forms.

Therapeutic Methods and Compositions

Any of the mutant IL-2 polypeptides and immunoconjugates provided hereinmay be used in therapeutic methods. Mutant IL-2 polypeptides andimmunoconjugates of the invention can be used as immunotherapeuticagents, for example in the treatment of cancers.

For use in therapeutic methods, mutant IL-2 polypeptides andimmunoconjugates of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

Mutant IL-2 polypeptides and immunoconjugates of the invention areuseful in treating disease states where stimulation of the immune systemof the host is beneficial, in particular conditions where an enhancedcellular immune response is desirable. These may include disease stateswhere the host immune response is insufficient or deficient. Diseasestates for which the mutant IL-2 polypeptides or immunoconjugates of theinvention can be administered comprise, for example, a tumor orinfection where a cellular immune response would be a critical mechanismfor specific immunity. Specific disease states for which IL-2 mutants ofthe present invention can be employed include cancer, for example renalcell carcinoma or melanoma; immune deficiency, specifically inHIV-positive patients, immunosuppressed patients, chronic infection andthe like. The mutant IL-2 polypeptides or immunoconjugates of theinvention may be administered per se or in any suitable pharmaceuticalcomposition.

In one aspect, mutant IL-2 polypeptides and immunoconjugates of theinvention for use as a medicament are provided. In further aspects,mutant IL-2 polypeptides and immunoconjugates of the invention for usein treating a disease are provided. In certain embodiments, mutant IL-2polypeptides and immunoconjugates of the invention for use in a methodof treatment are provided. In one embodiment, the invention provides amutant IL-2 polypeptide or an immunoconjugate as described herein foruse in the treatment of a disease in an individual in need thereof. Incertain embodiments, the invention provides a mutant IL-2 polypeptide oran immunoconjugate for use in a method of treating an individual havinga disease comprising administering to the individual a therapeuticallyeffective amount of the mutant IL-2 polypeptide or the immunoconjugate.In certain embodiments the disease to be treated is a proliferativedisorder. In a preferred embodiment the disease is cancer. In certainembodiments the method further comprises administering to the individuala therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In further embodiments, the invention provides amutant IL-2 polypeptide or an immunoconjugate for use in stimulating theimmune system. In certain embodiments, the invention provides a mutantIL-2 polypeptide or an immunoconjugate for use in a method ofstimulating the immune system in an individual comprising administeringto the individual an effective amount of the mutant IL-2 polypeptide orimmunoconjugate to stimulate the immune system. An “individual”according to any of the above embodiments is a mammal, preferably ahuman. “Stimulation of the immune system” according to any of the aboveembodiments may include any one or more of a general increase in immunefunction, an increase in T cell function, an increase in B cellfunction, a restoration of lymphocyte function, an increase in theexpression of IL-2 receptors, an increase in T cell responsiveness, anincrease in natural killer cell activity or lymphokine-activated killer(LAK) cell activity, and the like.

In a further aspect, the invention provides for the use of a mutant.IL-2 polypeptide or an immunoconjugate of the invention in themanufacture or preparation of a medicament for the treatment of adisease in an individual in need thereof. In one embodiment, themedicament is for use in a method of treating a disease comprisingadministering to an individual having the disease a therapeuticallyeffective amount of the medicament. In certain embodiments the diseaseto be treated is a proliferative disorder. In a preferred embodiment thedisease is cancer.

In one such embodiment, the method further comprises administering tothe individual a therapeutically effective amount of at least oneadditional therapeutic agent, e.g., an anti-cancer agent if the diseaseto be treated is cancer. In a further embodiment, the medicament is forstimulating the immune system. In a further embodiment, the medicamentis for use in a method of stimulating the immune system in an individualcomprising administering to the individual an amount effective of themedicament to stimulate the immune system. An “individual” according toany of the above embodiments may be a mammal, preferably a human.“Stimulation of the immune system” according to any of the aboveembodiments may include any one or more of a general increase in immunefunction, an increase in T cell function, an increase in B cellfunction, a restoration of lymphocyte function, an increase in theexpression of IL-2 receptors, an increase in T cell responsiveness, anincrease in natural killer cell activity or lymphokine-activated killer(LAK) cell activity, and the like.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to said individual atherapeutically effective amount of a mutant IL-2 polypeptide or animmunoconjugate of the invention. In one embodiment a composition isadministered to said individual, comprising the mutant IL-2 polypeptideor the immunoconjugate of the invention in a pharmaceutically acceptableform. In certain embodiments the disease to be treated is aproliferative disorder. In a preferred embodiment the disease is cancer.In certain embodiments the method further comprises administering to theindividual a therapeutically effective amount of at least one additionaltherapeutic agent, e.g., an anti-cancer agent if the disease to betreated is cancer. In a further aspect, the invention provides a methodfor stimulating the immune system in an individual, comprisingadministering to the individual an effective amount of a mutant IL-2polypeptide or an immunoconjugate to stimulate the immune system. An“individual” according to any of the above embodiments may be a mammal,preferably a human. “Stimulation of the immune system” according to anyof the above embodiments may include any one or more of a generalincrease in immune function, an increase in T cell function, an increasein B cell function, a restoration of lymphocyte function, an increase inthe expression of IL-2 receptors, an increase in T cell responsiveness,an increase in natural killer cell activity or lymphokine-activatedkiller (LAK) cell activity, and the like.

It is understood that any of the above therapeutic methods may becarried out using an immunoconjugate of the invention in place of or inaddition to a mutant IL-2 polypeptide.

In certain embodiments the disease to be treated is a proliferativedisorder, preferably cancer. Non-limiting examples of cancers includebladder cancer, brain cancer, head and neck cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, uterine cancer, cervicalcancer, endometrial cancer, esophageal cancer, colon cancer, colorectalcancer, rectal cancer, gastric cancer, prostate cancer, blood cancer,skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.Other cell proliferation disorders that can be treated using a mutantIL-2 polypeptide or an immunoconjugate of the present invention include,but are not limited to neoplasms located in the: abdomen, bone, breast,digestive system, liver, pancreas, peritoneum, endocrine glands(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid),eye, head and neck, nervous system (central and peripheral), lymphaticsystem, pelvic, skin, soft tissue, spleen, thoracic region, andurogenital system. Also included are pre-cancerous conditions or lesionsand cancer metastases. In certain embodiments the cancer is chosen fromthe group consisting of renal cell cancer, skin cancer, lung cancer,colorectal cancer, breast cancer, brain cancer, head and neck cancer.Similarly, other cell proliferation disorders can also be treated by themutant IL-2 polypeptides and immunoconjugates of the present invention.Examples of such cell proliferation disorders include, but are notlimited to: hypergammaglobulinemia, lymphoproliferative disorders,paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron'sMacroglobulinemia, Gaucher's Disease, histiocytosis, and any other cellproliferation disease, besides neoplasia, located in an organ systemlisted above. In another embodiment, the disease is related toautoimmunity, transplantation rejection, post-traumatic immune responsesand infectious diseases (e.g. HIV). More specifically, the mutant IL-2polypeptides and immunoconjugates may be used in eliminating cellsinvolved in immune cell-mediated disorders, including lymphoma;autoimmunity, transplantation rejection, graft-versus-host disease,ischemia and stroke. A skilled artisan readily recognizes that in manycases the mutant IL-2 polypeptides or immunoconjugates may not provide acure but may only provide partial benefit. In some embodiments, aphysiological change having some benefit is also consideredtherapeutically beneficial. Thus, in some embodiments, an amount ofmutant IL-2 polypeptide or immunoconjugate that provides a physiologicalchange is considered an “effective amount” or a “therapeuticallyeffective amount”. The subject, patient, or individual in need oftreatment is typically a mammal, more specifically a human.

The immunoconjugates of the invention are also useful as diagnosticreagents. The binding of an immunoconjugate to an antigenic determinantcan be readily detected by using a secondary antibody specific for theIL-2 polypeptide. In one embodiment, the secondary antibody and theimmunoconjugate facilitate the detection of binding of theimmunoconjugate to an antigenic determinant located on a cell or tissuesurface.

In some embodiments, an effective amount of the mutant IL-2 polypeptidesor immunoconjugates of the invention is administered to a cell. In otherembodiments, a therapeutically effective amount of the mutant IL-2polypeptides or immunoconjugates of the invention is administered to anindividual for the treatment of disease.

For the prevention or treatment of disease, the appropriate dosage of amutant IL-2 polypeptide or immunoconjugate of the invention (when usedalone or in combination with one or more other additional therapeuticagents) will depend on the type of disease to be treated, the route ofadministration, the body weight of the patient, the type of polypeptide(e.g. unconjugated IL-2 or immunoconjugate), the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous or concurrent therapeutic interventions,the patient's clinical history and response to the mutant IL-2polypeptide or immunoconjugate, and the discretion of the attendingphysician. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

A single administration of unconjugated IL-2 can range from about 50,000IU/kg to about 1,000,000 IU/kg or more, more typically about 600,000IU/kg of IL-2. This may be repeated several times a day (e.g. 2-3×), forseveral days (e.g. about 3-5 consecutive days) and then may be repeatedone or more times following a period of rest (e.g., about 7-14 days).Thus, a therapeutically effective amount may comprise only a singleadministration or many administrations over a period of time (e.g. about20-30 individual administrations of about 600,000 IU/kg of IL-2 eachgiven over about a 10-20 day period). When administered in the form ofan immunoconjugate, a therapeutically effective of the mutant IL-2polypeptide may be lower than for unconjugated mutant IL-2 polypeptide.

Similarly, the immunoconjugate is suitably administered to the patientat one time or over a series of treatments. Depending on the type andseverity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10mg/kg) of immunoconjugate can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the immunoconjugate would be in therange from about 0.005 mg/kg to about 10 mg/kg. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, or e.g. about six doses of the immunoconjugate). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. The progressof this therapy is easily monitored by conventional techniques andassays.

The mutant IL-2 polypeptides and immunoconjugates of the invention willgenerally be used in an amount effective to achieve the intendedpurpose. For use to treat or prevent a disease condition, the mutantIL-2 polypeptides and immunoconjugates of the invention, orpharmaceutical compositions thereof, are administered or applied in atherapeutically effective amount. Determination of a therapeuticallyeffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose can beestimated initially from in vitro assays, such as cell culture assays. Adose can then be formulated in animal models to achieve a circulatingconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animalmodels, using techniques that are well known in the art. One havingordinary skill in the art could readily optimize administration tohumans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the mutant IL-2 polypeptides or immunoconjugates whichare sufficient to maintain therapeutic effect. Usual patient dosages foradministration by injection range from about 0.1 to 50 mg/kg/day,typically from about 0.5 to 1 mg/kg/day. Therapeutically effectiveplasma levels may be achieved by administering multiple doses each day.Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the immunoconjugates may not be related to plasmaconcentration. One having skill in the art will be able to optimizetherapeutically effective local dosages without undue experimentation.

A therapeutically effective dose of the mutant IL-2 polypeptides orimmunoconjugates described herein will generally provide therapeuticbenefit without causing substantial toxicity. Toxicity and therapeuticefficacy of an IL-2 mutant or immunoconjugate can be determined bystandard pharmaceutical procedures in cell culture or experimentalanimals (see, e.g. Examples 8 and 9). Cell culture assays and animalstudies can be used to determine the LD₅₀ (the dose lethal to 50% of apopulation) and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. IL-2mutants and immunoconjugates that exhibit large therapeutic indices arepreferred. In one embodiment, the mutant IL-2 polypeptide or theimmunoconjugate according to the present invention exhibits a hightherapeutic index. The data obtained from cell culture assays and animalstudies can be used in formulating a range of dosages suitable for usein humans. The dosage lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon a variety of factors,e.g., the dosage form employed, the route of administration utilized,the condition of the subject, and the like. The exact formulation, routeof administration and dosage can be chosen by the individual physicianin view of the patient's condition. (See, e.g., Fingl et al., 1975, In:The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, incorporatedherein by reference in its entirety)

The attending physician for patients treated with IL-2 mutants orimmunoconjugates of the invention would know how and when to terminate,interrupt, or adjust administration due to toxicity, organ dysfunction,and the like. Conversely, the attending physician would also know toadjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administered dose inthe management of the disorder of interest will vary with the severityof the condition to be treated, with the route of administration, andthe like. The severity of the condition may, for example, be evaluated,in part, by standard prognostic evaluation methods. Further, the doseand perhaps dose frequency will also vary according to the age, bodyweight, and response of the individual patient.

The maximum therapeutic dose of a mutant IL-2 polypeptide orimmunoconjugate comprising said polypeptide may be increased from thoseused for wild-type IL-2 or an immunoconjugate comprising wild-type IL-2,respectively.

Other Agents and Treatments

The mutant IL-2 polypeptides and the immunoconjugates according to theinvention may be administered in combination with one or more otheragents in therapy. For instance, a mutant IL-2 polypeptide orimmunoconjugate of the invention may be co-administered with at leastone additional therapeutic agent. The term “therapeutic agent”encompasses any agent administered to treat a symptom or disease in anindividual in need of such treatment. Such additional therapeutic agentmay comprise any active ingredients suitable for the particularindication being treated, preferably those with complementary activitiesthat do not adversely affect each other. In certain embodiments, anadditional therapeutic agent is an immunomodulatory agent, a cytostaticagent, an inhibitor of cell adhesion, a cytotoxic agent, an activator ofcell apoptosis, or an agent that increases the sensitivity of cells toapoptotic inducers. In a particular embodiment, the additionaltherapeutic agent is an anti-cancer agent, for example a microtubuledisruptor, an antimetabolite, a topoisomerase inhibitor, a DNAintercalator, an alkylating agent, a hormonal therapy, a kinaseinhibitor, a receptor antagonist, an activator of tumor cell apoptosis,or an antiangiogenic agent.

Such other agents are suitably present in combination in amounts thatare effective for the purpose intended. The effective amount of suchother agents depends on the amount of mutant IL-2 polypeptide orimmunoconjugate used, the type of disorder or treatment, and otherfactors discussed above. The mutant IL-2 polypeptides andimmunoconjugates are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate compositions), and separate administration, in which case,administration of the mutant IL-2 polypeptide or immunoconjugate of theinvention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.Mutant IL-2 polypeptides and immunoconjugates of the invention can alsobe used in combination with radiation therapy.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a mutant IL-2 polypeptide of the invention. The label orpackage insert indicates that the composition is used for treating thecondition of choice. Moreover, the article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises a mutant IL-2 polypeptide of the invention; and(b) a second container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The article of manufacture in this embodiment of the inventionmay further comprise a package insert indicating that the compositionscan be used to treat a particular condition. Alternatively, oradditionally, the article of manufacture may further comprise a second(or third) container comprising a pharmaceutically-acceptable buffer,such as bacteriostatic water for injection (BWFI), phosphate-bufferedsaline, Ringer's solution and dextrose solution. It may further includeother materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto a mutant IL-2 polypeptide.

SHORT DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of the Fab-IL-2-Fab (A) and IgG-IL-2(B) immunoconjugate formats, comprising mutant IL-2 polypeptide.

FIG. 2. Purification of the naked IL-2 wild-type construct. (A)Chromatogram of the His tag purification for the wild-type naked IL-2;(B) SDS PAGE of purified protein (8-12% Bis-Tris (NuPage, Invitrogen),MES running buffer).

FIG. 3. Purification of the naked IL-2 wild-type construct. (A)Chromatogram of the size exclusion chromatography for the wild-typeIL-2; (B) SDS PAGE of purified protein (8-12% Bis-Tris (NuPage,Invitrogen), MES running buffer).

FIG. 4. Analytical size exclusion chromatography for the wild-type IL-2as determined on a Superdex 75, 10/300 GL. Pool 1 comprises 74% of the23 kDa species and 26% of the 20 kDa species, Pool 2 comprises 40% ofthe 22 kDa species and 60% of the 20 kDa species.

FIG. 5. Purification of the naked IL-2 quadruple mutant construct. (A)Chromatogram of the His tag purification for the IL-2 quadruple mutant;(B) SDS PAGE of purified protein (8-12% Bis-Tris (NuPage, Invitrogen),MES running buffer).

FIG. 6. Purification of the naked IL-2 quadruple mutant construct. (A)Chromatogram of the size exclusion chromatography for the IL-2 quadruplemutant; (B) SDS PAGE of purified protein (8-12% Bis-Tris (NuPage,Invitrogen), MES running buffer).

FIG. 7. Analytical size exclusion chromatography for the IL-2 quadruplemutant as determined on a Superdex 75, 10/300 GL (Pool 2, 20 kDa).

FIG. 8. Simultaneous binding to IL-2R and human FAP by FAP-targeted29B11-based Fab-IL-2-Fab comprising wild-type or quadruple mutant IL-2.(A) Setup of the SPR assay; (B) SPR sensorgram.

FIG. 9. Induction of IFN-γ release by NK92 cells by FAP-targeted4G8-based Fab-IL-2-Fab comprising wild-type or mutant IL-2, compared toProleukin, in solution.

FIG. 10. Induction of proliferation of isolated NK cells (bottom) byFAP-targeted 4G8-based Fab-IL-2-Fab comprising wild-type or mutant IL-2,compared to Proleukin, in solution.

FIG. 11. Induction of proliferation of activated CD3⁺ T cells byFAP-targeted 4G8-based Fab-IL-2-Fab comprising wild-type of mutant IL-2,compared to Proleukin, in solution.

FIG. 12. Induction of activation induced cell death (AICD) ofover-stimulated T cells by FAP-targeted 4G8-based Fab-IL-2-Fabcomprising wild-type or mutant IL-2, compared to Proleukin, in solution.

FIG. 13. Phospho-STAT5 FACS assay in solution with FAP-targeted4G8-based Fab-IL-2-Fab comprising wild-type or quadruple mutant IL-2,compared to Proleukin, in solution. (A) regulatory T cells(CD4⁺CD25⁺FOXP3⁺); (B) CD8⁺ T cells (CD3⁺CD8⁺); (C) CD4⁺ T cells(CD4⁺CD25⁻CD127⁺); (D) NK cells (CD3⁻CD56⁺).

FIG. 14. Purification of the FAP-targeted 28H1-based Fab-IL-2 qm-Fabimmunoconjugate. (A) Elution profile of Protein G column. (B) Elutionprofile of Superdex 200 size exclusion column. (C) Novex Tris-Glycine4-20% SDS-PAGE of the end-product with non-reduced and reduced sample.

FIG. 15. Purification of the 4G8-based FAP-targeted Fab-IL-2 qm-Fabimmunoconjugate. (A) Elution profile of Protein A column. (B) Elutionprofile of Superdex 200 size exclusion column. (C) NuPAGE Novex Bis-TrisMini Gel (Invitrogen), MOPS running buffer of the end-product withnon-reduced and reduced sample.

FIG. 16. Purification of the MHLG1 KV9 MCSP-targeted Fab-IL2QM-Fabimmunoconjugate. (A) Elution profile of Protein A column, B) Elutionprofile of Superdex 200 size exclusion column. C) NuPAGE Novex Bis-TrisMini Gel, Invitrogen, MOPS running buffer of the end-product withnon-reduced and reduced sample.

FIG. 17. Target binding of Fab-IL-2-Fab constructs on HEK 293-human FAPcells.

FIG. 18. Target binding of Fab-IL-2-Fab constructs on HEK 293-human FAPcells.

FIG. 19. Binding specificity of Fab-IL-2-Fab constructs as determined onHEK 293-human DPPIV and HEK 293 mock-transfected cells. Binding of aspecific DPPIV (CD26) antibody is shown on the right.

FIG. 20. Analysis of FAP internalization upon binding of Fab-IL-2-Fabconstructs to FAP on GM0S389 fibroblasts.

FIG. 21. IL-2 induced IFN-γ release by NK92 cells in solution.

FIG. 22. IL-2 induced IFN-γ release by NK92 cells in solution.

FIG. 23. IL-2 induced proliferation of NK92 cells in solution.

FIG. 24. Assessment of Fab-IL-2-Fab clones 28H1 vs. 29B11 vs. 4G8 inSTAT5 phosphorylation assay with PBMCs in solution. (A) NK cells(CD3⁻CD56⁺); (B) CD8⁺ T cells (CD3⁺CD8⁺); (C) CD4⁺ T cells(CD3⁺CD4⁺CD25⁻CD127⁺); (D) regulatory T cells (CD4⁺CD25⁺FOXP3⁺).

FIG. 25. Efficacy of the FAP-targeted 4G8 Fab-IL-2 wt-Fab and 4G8Fab-IL-2 qm-Fab immunoconjugates in the human renal cell adenocarcinomacell line ACHN.

FIG. 26. Efficacy of the FAP-targeted 4G8 FAP-IL-2 qm-Fab and 28H1Fab-IL-2 qm-Fab immunoconjugates in the mouse Lewis lung carcinoma cellline LLC1.

FIG. 27. Efficacy of the FAP-targeted 28H1 Fab-IL-2 wt-Fab and 28H1Fab-IL-2 qm-Fab immunoconjugates in the mouse Lewis lung carcinoma cellline LLC1.

FIG. 28. Low magnification (100×) of lungs of mice treated with vehiclecontrol (A) or 9 μg/g wt IL-2 (B) or qm IL-2 (C). Lungs of mice treatedwith 9 μg/g wt IL-2 show vasocentric mononuclear infiltrate that hasmoved into the alveolar spaces. Edema and hemorrhage is also present.Marginal infiltrate is noted in the mice treated with qm IL-2 around fewvessels.

FIG. 29. Higher magnification (200×) of lungs shown in FIG. 28.Margination and infiltration of mononuclear cells in and around bloodvessels is more severe in mice treated with wt IL-2-(A) than in micetreated with qm IL-2 (B and C).

FIG. 30. Low magnification (100×) of livers of mice treated with vehiclecontrol (A) or 9 μg/g wt IL-2 (B) or qm IL-2 (C). Vasocentricinfiltration is seen in mice treated with wt IL-2.

FIG. 31. IFN-γ secretion by NK92 cells upon incubation with differentIL-2 wild-type (wt) and quadruple mutant (qm) preparations for 24 (A) or48 hours (B).

FIG. 32. Proliferation of NK92 cells upon incubation with different IL-2wild-type (wt) and quadruple mutant (qm) preparations for 48 hours.

FIG. 33. Proliferation of NK92 cells upon incubation with different IL-2wild-type (wt) and quadruple mutant (qm) preparations for 48 hours.

FIG. 34. Proliferation of NK cells upon incubation with differentFAP-targeted 28H1 IL-2 immunoconjugates or Proleukin for 4 (A), 5 (B) or6 (C) days.

FIG. 35. Proliferation of CD4 T-cells upon incubation with differentFAP-targeted 28H1 IL-2 immunoconjugates or Proleukin for 4 (A), 5 (B) or6 (C) days.

FIG. 36. Proliferation of CD8 T-cells upon incubation with differentFAP-targeted 28H1 IL-2 immunoconjugates or Proleukin for 4 (A), 5 (B) or6 (C) days.

FIG. 37. Proliferation of NK cells (A), CD4 T-cells (B) and CD8 T-cells(C) upon incubation with different IL-2 immunoconjugates or Proleukinfor 6 days.

FIG. 38. STAT phosphorylation in NK cells (A), CD8 T-cells (B), CD4T-cells (C) and regulatory T-cells (D) after 30 min incubation withProleukin, in-house produced wild-type IL-2 and quadruple mutant IL-2.

FIG. 39. STAT phosphorylation in NK cells (A), CD8 T-cells (B), CD4T-cells (C) and regulatory T-cells (D) after 30 min incubation withProleukin, IgG-IL-2 comprising wild-type IL-2 or IgG-IL-2 comprisingquadruple mutant IL-2.

FIG. 40. Survival of Black 6 mice after administration (once daily forseven days) of different doses of IL-2 immunoconjugates comprisingwild-type or quadruple mutant IL-2.

FIG. 41. Serum concentrations of IL-2 immunoconjugates after a singlei.v. administration of FAP-targeted (A) and untargeted (B) IgG-IL-2constructs comprising either wild-type (wt) or quadruple mutant (qm)IL-2.

FIG. 42. Serum concentrations of IL-2 immunoconjugates after a singlei.v. administration of untargeted Fab-IL-2-Fab constructs comprisingeither wild-type (wt) or quadruple mutant (qm) IL-2.

FIG. 43. Purification of quadruple mutant IL-2. (A) Immobilized metalion chromatography; (B) size exclusion chromatography; (C) SDS PAGEunder non-reducing conditions (NuPAGE Novex Bis-Tris gel (Invitrogen),MES running buffer); (D) analytical size exclusion chromatography(Superdex 75 10/300 GL).

FIG. 44. Proliferation of pre-activated CD8 (A) and CD4 (B) T cellsafter six days incubation with different IL-2 immunoconjugates.

FIG. 45. Activation induced cell death of CD3⁺ T cells after six daysincubation with different IL-2 immunoconjugates and overnight treatmentwith anti-Fas antibody.

FIG. 46. Purification of FAP-targeted 4G8-based IgG-IL-2 quadruplemutant (qm) immunoconjugate. A) Elution profile of the Protein Aaffinity chromatography step. B) Elution profile of the size exclusionchromatography step. C) Analytical SDS-PAGE (NuPAGE Novex Bis-Tris MiniGel, Invitrogen, MOPS running buffer) of the final product. D)Analytical size exclusion chromatography of the final product on aSuperdex 200 column (97% monomer content).

FIG. 47. Purification of FAP-targeted 28H1-based IgG-IL-2 qmimmunoconjugate. A) Elution profile of the Protein A affinitychromatography step. B) Elution profile of the size exclusionchromatography step. C) Analytical SDS-PAGE (reduced: NuPAGE NovexBis-Tris Mini Gel, Invitrogen, MOPS running buffer; non-reduced: NuPAGETris-Acetate, Invitrogen, Tris-Acetate running buffer) of the finalproduct. D) Analytical size exclusion chromatography of the finalproduct on a Superdex 200 column (100% monomer content).

FIG. 48. Binding of FAP-targeted 4G8-based IgG-IL-2 qm immunoconjugateto human FAP expressed on stably transfected HEK 293 cells as measuredby FACS, compared to the corresponding Fab-IL-2 qm-Fab construct.

FIG. 49. Interferon (IFN)-γ release on NK92 cells induced byFAP-targeted 4G8-based IgG-IL-2 qm immunoconjugate in solution, comparedto the 28H1-based Fab-IL-2 qm-Fab construct.

FIG. 50. Detection of phosphorylated STAT5 by FACS in different celltypes after stimulation for 20 min with FAP-targeted 4G8-based IgG-IL-2qm immunoconjugate in solution, compared to the 28H1-based Fab-IL-2-Faband Fab-IL-2 qm-Fab constructs as well as Proleukin. A) NK cells(CD3⁻CD56⁺); B) CD8⁺ T cells (CD3⁺CD8⁺); C) CD4⁺ T cells(CD3⁺CD4⁺CD25⁻CD127⁺); D) regulatory T cells (CD4⁺CD25⁺FOXP3⁺).

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1

General Methods

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook etal., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. General information regarding the nucleotide sequences ofhuman immunoglobulins light and heavy chains is given in: Kabat, E. A.et al., (1991) Sequences of Proteins of Immunological Interest, FifthEd., NIH Publication No 91-3242.

DNA Sequencing

DNA sequences were determined by double strand sequencing.

Gene Synthesis

Desired gene segments where required were either generated by PCR usingappropriate templates or were synthesized by Geneart AG (Regensburg,Germany) from synthetic oligonucleotides and PCR products by automatedgene synthesis. In cases where no exact gene sequence was available,oligonucleotide primers were designed based on sequences from closesthomologues and the genes were isolated by RT-PCR from RNA originatingfrom the appropriate tissue. The gene segments flanked by singularrestriction endonuclease cleavage sites were cloned into standardcloning/sequencing vectors. The plasmid DNA was purified fromtransformed bacteria and concentration determined by UV spectroscopy.The DNA sequence of the subcloned gene fragments was confirmed by DNAsequencing. Gene segments were designed with suitable restriction sitesto allow sub-cloning into the respective expression vectors. Allconstructs were designed with a 5′-end DNA sequence coding for a leaderpeptide which targets proteins for secretion in eukaryotic cells. SEQ IDNOs 263-273 give exemplary leader peptides and polynucleotide sequencesencoding them.

Preparation of IL-2R βγ Subunit-Fc Fusions and IL-2R α Subunit Fc Fusion

To study IL-2 receptor binding affinity, a tool was generated thatallowed for the expression of a heterodimeric IL-2 receptor, theβ-subunit of the IL-2 receptor was fused to an Fc molecule that wasengineered to heterodimerize (Fc(hole)) (see SEQ ID NOs 274 and 275)using the “knobs-into-holes” technology (Merchant et al., Nat Biotech.16, 677-681 (1998)). The γ-subunit of the IL-2 receptor was then fusedto the Fc(knob) variant (see SEQ ID NOs 276 and 277), whichheterodimerized with Fc(hole). This heterodimeric Fc-fusion protein wasthen used as a substrate for analyzing the IL2/IL-2 receptorinteraction. The IL-2R α-subunit was expressed as monomeric chain withan AcTev cleavage site and an Avi His tag (SEQ ID NOs 278 and 279). Therespective IL-2R subunits were transiently expressed in HEK EBNA 293with serum for the IL-2R βγ subunit construct and without serum for theα-subunit construct. The IL-2R βγ subunit construct was purified onprotein A (GE Healthcare), followed by size exclusion chromatography (GEHealthcare, Superdex 200). The IL-2R α-subunit was purified via His tagon a NiNTA column (Qiagen) followed by size exclusion chromatography (GEHealthcare, Superdex 75).

Preparation of Immunoconjugates

Details about the preparation and purification of Fab-IL-2-Fabimmunoconjugates, including generation and affinity maturation ofantigen binding moieties can be found in the Examples appended to PCTpublication no. WO 2011/020783, which is incorporated herein byreference in its entirety. As described therein, various antigen bindingdomains directed to FAP have been generated by phage display, includingthe ones designated 4G8, 3F2, 28H1, 29B11, 14B3, and 4B9 used in thefollowing examples. Clone 28H1 is an affinity matured antibody based onparental clone 4G8, while clones 29B11, 14B3 and 4B9 are affinitymatured antibodies based on parental clone 3F2. The antigen bindingdomain designated MHLG1 KV9 used herein is directed to MCSP.

The sequences of immunoconjugates comprising wild-type IL-2 that wereused in the following examples can also be found in PCT publication no.WO 2011/020783. The sequences corresponding to the immunoconjugatescomprising quadruple mutant IL-2 that were used in the followingexamples are: 4G8: SEQ ID NOs 211 and 233; 3F2: SEQ ID NOs 209 and 231;28H1: SEQ ID NOs 219 and 233; 29B11: SEQ ID NOs 221 and 231; 14B3: SEQID NOs 229 and 231; 4B9: SEQ ID NOs 227 and 231; MHLG1-KV9: SEQ ID NOs253 and 255. The DNA sequences were generated by gene synthesis and/orclassical molecular biology techniques and subcloned into mammalianexpression vectors (one for the light chain and one for the heavychain/IL-2 fusion protein) under the control of an MPSV promoter andupstream of a synthetic polyA site, each vector carrying an EBV OriPsequence. Immunoconjugates as applied in the examples below wereproduced by co-transfecting exponentially growing HEK293-EBNA cells withthe mammalian expression vectors using calcium phosphate-transfection.Alternatively, HEK293 cells growing in suspension were transfected bypolyethylenimine (PEI) with the respective expression vectors.Alternatively, stably transfected CHO cell pools or CHO cell clones wereused for production in serum-free media. While 4G8-based FAP-targetedFab-IL-2-Fab constructs comprising wild-type or (quadruple) mutant IL-2can be purified by affinity chromatography using a protein A matrix,affinity matured 28H1-based FAP-targeted Fab-IL-2-Fab constructs werepurified by affinity chromatography on a protein G matrix in smallscale.

Briefly, FAP-targeted 28H1 Fab-IL-2-Fab, comprising wild-type or(quadruple) mutant IL-2, was purified from cell supernatants by oneaffinity step (protein G) followed by size exclusion chromatography(Superdex 200, GE Healthcare). The protein G column was equilibrated in20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, supernatant wasloaded, and the column was washed with 20 mM sodium phosphate, 20 mMsodium citrate pH 7.5. Fab-IL-2-Fab was eluted with 8.8 mM formic acidpH 3. The eluted fractions were pooled and polished by size exclusionchromatography in the final formulation buffer: 25 mM potassiumphosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7. Exemplaryresults from purification and analytics are given below.

FAP-targeted 3F2 Fab-IL-2-Fab or 4G8 Fab-IL-2-Fab, comprising wild-typeor (quadruple) mutant IL-2, were purified by a similar method composedof one affinity step using protein A followed by size exclusionchromatography (Superdex 200, GE Healthcare). The protein A column wasequilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5,supernatant was loaded, and the column was washed with 20 mM sodiumphosphate, 20 mM sodium citrate, 500 mM sodium chloride pH 7.5, followedby a wash with 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mMsodium chloride pH 5.45. A third wash with 10 mM MES, 50 mM sodiumchloride pH 5 was optionally performed. Fab-IL-2-Fab was eluted with 20mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3. Theeluted fractions were pooled and polished by size exclusionchromatography in the final formulation buffer 25 mM potassiumphosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7. Exemplarydetailed purification procedures and results are given for selectedconstructs below.

FAP-targeted IgG-IL-2 qm fusion proteins were generated based on theFAP-antibodies 4G8, 4B9 and 28H1, wherein one single IL-2 quadruplemutant (qm) was fused to the C-terminus of one heterodimeric heavy chainas shown in FIG. 1B. Targeting to the tumor stroma where FAP isselectively expressed is achieved via the bivalent antibody Fab region(avidity effect).

Heterodimerization resulting in the presence of a single IL-2 quadruplemutant is achieved by application of the knob-into-hole technology. Inorder to minimize the generation of homodimeric IgG-cytokine fusions thecytokine was fused to the C-terminus (with deletion of the C-terminalLys residue) of the knob-containing IgG heavy chain via a G₄-(SG₄)₂- or(G₄S)₃-linker. The antibody-cytokine fusion has IgG-like properties. Toreduce FcγR binding/effector function and prevent FcR co-activation,P329G L234A L235A (LALA) mutations were introduced in the Fc domain. Thesequences of these immunoconjugates are given in SEQ ID NOs 297, 299 and233 (28H1), SEQ ID NOs 301, 303 and 231 (4B9), and SEQ ID NOs 315, 317and 233 (4G8)). In addition, a CEA-targeted IgG-IL-2 qm fusion proteinand a control DP47GS non-targeted IgG-IL-2 qm fusion protein wherein theIgG does not bind to a specified target was generated. The sequences ofthese immunoconjugates are given in SEQ ID NOs 305, 307 and 309(DP47GS), and SEQ ID NOs 319, 321 and 323 (CH1A1A).

The IgG-IL-2 constructs were generated by transient expression in HEK293EBNA cells and purified essentially as described above for theFab-IL-2-Fab constructs. Briefly, IgG-IL-2 fusion proteins were purifiedby one affinity step with protein A (HiTrap ProtA, GE Healthcare)equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5.After loading of the supernatant, the column was first washed with 20 mMsodium phosphate, 20 mM sodium citrate, pH 7.5 and subsequently washedwith 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodiumchloride, pH 5.45. The IgG-cytokine fusion protein was eluted with 20 mMsodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3. Fractionswere neutralized and pooled and purified by size exclusionchromatography (HiLoad 16/60 Superdex 200, GE Healthcare) in finalformulation buffer: 25 mM potassium phosphate, 125 mM sodium chloride,100 mM glycine pH 6.7. Exemplary detailed purification procedures andresults are given for selected constructs below. The proteinconcentration of purified protein samples was determined by measuringthe optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of immunoconjugates were analyzed by SDS-PAGE inthe presence and absence of a reducing agent (5 mM 1,4-dithiothreitol)and stained with Coomassie blue (SimpleBlue™ SafeStain, Invitrogen). TheNuPAGE® Pre-Cast gel system (Invitrogen) was used according to themanufacturer's instructions (4-20% Tris-glycine-gels or 3-12% Bis-Tris).The aggregate content of immunoconjugate samples was analyzed using aSuperdex 200 10/300GL analytical size-exclusion column (GE Healthcare)in 2 mM MOPS, 150 mM NaCl, 0.02% NaN₃, pH 7.3 running buffer at 25° C.

FAP Binding Affinity

The FAP binding activity of the cleaved Fab fragments used in theseexamples as antigen binding, moieties was determined by surface plasmonresonance (SPR) on a Biacore machine. Briefly, an anti-His antibody(Penta-His, Qiagen 34660) was immobilized on CM5 chips to capture 10 nMhuman, murine or cynomolgus FAP-His (20 s). Temperature was 25° C. andHBS-EP was used as buffer. Fab analyte concentration was 100 nM down to0.41 nM (duplicates) at a flow rate of 50 μl/min (association: 300 s,dissociation: 600 s (4B9, 14B3, 29B11, 3F2) or 1200 s (28H1, 4G8),regeneration: 60 s 10 mM glycine pH 2). Fitting was performed based on a1:1 binding model, RI=0, Rmax=local (because of capture format). Table 2gives the monovalent affinities as determined by SPR.

TABLE 2 Affinity (K_(D)) of FAP-targeted Fab fragments to FAP asdetermined by SPR. K_(D) in nM Human FAP Cynomolgus FAP Murine FAP 4B9Fab 0.3 0.23 5 0.31 0.24 5.2 14B3 Fab 0.47 0.61 4.7 0.47 0.59 4.7 29B11Fab 0.19 0.21 1.3 0.19 0.2 1.2 3F2 Fab 6 4.7 8.9 6 5.3 9.5 28H1 Fab 2.63.7 0.13 2.6 3.7 0.18 4G8 Fab 53 (48 steady state) 33 (33 steady state)0.07 51 (48 steady state) 35 (34 steady state) 0.07

Biological Activity Assays with Targeted IL-2 Immunoconjugates

The biological activity of FAP- or MCSP-targeted Fab-IL-2-Fabimmunoconjugates and of FAP-targeted IgG-IL-2 immunoconjugates,comprising wild-type or (quadruple) mutant IL-2, was investigated inseveral cellular assays in comparison to commercially available IL-2(Proleukin, Novartis, Chiron).

IFN-γ Release by NK Cells (in Solution)

IL-2 starved NK92 cells (100000 cells/well in 96-U-well plate) wereincubated with different concentrations of IL-2 immunoconjugates,comprising wild-type or (quadruple) mutant IL-2, for 24 h in NK medium(MEM alpha from Invitrogen (#22561-021) supplemented with 10% FCS, 10%horse serum, 0.1 mM 2-mercaptoethanol, 0.2 mM inositol and 0.02 mM folicacid). Supernatants were harvested and the IFN-γ release was analysedusing the anti-human IFN-γ ELISA Kit II from Becton Dickinson (#550612).Proleukin (Novartis) served as positive control for IL-2-mediatedactivation of the cells.

NK Cell Proliferation

Blood from healthy volunteers was taken in heparin-containing syringesand PBMCs were isolated. Untouched human NK cells were isolated from thePBMCs using the Human NK Cell Isolation Kit II from Miltenyi Biotec(#130-091-152). The CD25 expression of the cells was checked by flowcytometry. For proliferation assays, 20000 isolated human NK cells wereincubated for 2 days in a humidified incubator at 37° C., 5% CO₂ in thepresence of different IL-2 immunoconjugates, comprising wild-type or(quadruple) mutant IL-2. Proleukin (Novartis) served as control. After 2days, the ATP content of the cell lysates was measured using theCellTiter-Glo Luminescent Cell Viability Assay from Promega(#G7571/2/3). The percentage of growth was calculated setting thehighest Proleukin concentration to 100% proliferation and untreatedcells without IL-2 stimulus to 0% proliferation.

STAT5 Phosphorylation Assay

Blood from healthy volunteers was taken in heparin-containing syringesand PBMCs were isolated. PBMCs were treated with IL-2 immunoconjugates,comprising wild-type or (quadruple) mutant IL-2, at the indicatedconcentrations or with Proleukin (Novartis) as control. After 20 minincubation at 37° C., PBMCs were fixed with pre-warmed Cytofix buffer(Becton Dickinson #554655) for 10 min at 37° C., followed bypermeabilization with Phosflow Perm Buffer III (Becton Dickinson#558050) for 30 min at 4° C. Cells were washed twice with PBS containing0.1% BSA before FACS staining was performed using mixtures of flowcytometry antibodies for detection of different cell populations andphosphorylation of STAT5. Samples were analysed using a FACSCantoII withHTS from Becton Dickinson.

NK cells were defined as CD3⁻CD56⁺, CD8 positive T cells were defined asCD3⁺CD8⁺, CD4 positive T cells were defined as CD4⁺CD25⁻CD127⁺ andT_(reg) cells were defined as CD4⁺CD25⁺FoxP3⁺.

Proliferation and AICD of T Cells

Blood from healthy volunteers was taken in heparin-containing syringesand PBMCs were isolated. Untouched T cells were isolated using the Pan TCell Isolation Kit II from Miltenyi Biotec (#130-091-156). T cells werepre-stimulated with 1 μg/ml PHA-M (Sigma Aldrich #L8902) for 16 h beforeadding Proleukin or Fab-IL-2-Fab immunoconjugates, comprising wild-typeor (quadruple) mutant IL-2, to the washed cells for another 5 days.After 5 days, the ATP content of the cell lysates was measured using theCellTiter-Glo Luminescent Cell Viability Assay from Promega(#G7571/2/3). The relative proliferation was calculated setting thehighest Proleukin concentration to 100% proliferation.

Phosphatidylserine (PS) exposure and cell death of T cells were assayedby flow cytometric analysis (FACSCantoII, BD Biosciences) of annexin V(Annexin-V-FLUOS Staining Kit, Roche Applied Science) and propidiumiodide (PI)-stained cells. To induce activation-induced cell death(AICD); the T cells were treated with an apoptosis-inducing anti-Fasantibody (Millipore clone Ch11) for 16 h after the 16 h PHA-M and 5 daystreatment with Fab-IL-2-Fab immunoconjugates. Annexin V staining wasperformed according to the manufacturer's instructions. Briefly, cellswere washed with Ann-V Binding Buffer (Ix stock: 0.01 M Hepes/NaOHpH7.4, 0.14 M NaCl, 2.5 mM CaCl₂) and stained for 15 min at RT in thedark with Annexin V FITC (Roche); Cells were washed again inAnn-V-Binding buffer before addition of 200 μl/well Ann-V-Binding Buffercontaining PI (0.3 μg/ml). The cells were analysed immediately by flowcytometry.

Binding to FAP Expressing Cells

Binding of FAP-targeted IgG-IL-2 qm and Fab-IL-2 qm-Fab immunoconjugatesto human FAP expressed on stably transfected HEK293 cells was measuredby FACS. Briefly, 250 000 cells per well were incubated with theindicated concentration of the immunoconjugates in a round-bottom96-well plate, incubated for 30 min at 4° C., and washed once withPBS/0.1% BSA. Bound immunoconjugates were detected after incubation for30 min at 4° C. with FITC-conjugated AffiniPure F(ab′)2 Fragment goatanti-human F(ab′)2 Specific (Jackson Immuno Research Lab #109-096-097,working solution: 1:20 diluted in PBS/0.1% BSA, freshly prepared) usinga FACS CantoII (Software FACS Diva).

Analysis of FAP Internalization Upon Binding by FACS

For several FAP antibodies known in the art it is described that theyinduce FAP internalization upon binding (described e.g. in Baum et al.,J Drug Target 15, 399-406 (2007); Bauer at al., Journal of ClinicalOncology, 2010 ASCO Annual Meeting Proceedings (Post-Meeting Edition),vol. 28 (May 20 Supplement), abstract no. 13062 (2010); Ostermann etal., Clin Cancer Res 14, 4584-4592 (2008)). Thus, we analyzed theinternalization properties of our Fab-IL-2-Fab immunoconjugates.Briefly, GM05389 cells (human lung fibroblasts,) cultured in EMEM mediumwith 15% FCS, were detached, washed, counted, checked for viability andseeded at a density of 2×10⁵ cells/well in 12-well plates. The next day,FAP-targeted Fab-IL-2-Fab immunoconjugates were diluted in cold mediumand allowed to bind to cell surface for 30 min on ice. The excess ofunbound antibody was washed away using cold PBS and cells were furtherincubated in 0.5 ml complete pre-warmed medium at 37° C. for theindicated time periods. When the different time points were reached,cells were transferred on ice, washed once with cold PBS and incubatedwith the secondary antibody (FITC-conjugated AffiniPure F(ab′)2 Fragmentgoat anti-human F(ab′)2 specific, Jackson Immuno Research Lab#109-096-097, 1:20 dilution) for 30 min at 4° C. Cells were then washedtwice with PBS/0.1% BSA, transferred to a 96-well plate, centrifuged for4 min at 4° C., 400×g and cell pellets were resuspended by vortexing.Cells were fixed using 100 μl 2% PFA. For FACS measurement, cells werere-suspended in 200 μl/sample PBS/0.1% BSA and measured with the plateprotocol in FACS CantoII (Software FACS Diva).

Example 2

We designed mutated versions of IL-2 that comprised one or more of thefollowing mutations (compared to the wild-type IL-2 sequence shown inSEQ ID NO: 1):

-   -   1. T3A—knockout of predicted O-glycosylation site    -   2. F42A—knockout of IL-2/IL-2R α interaction    -   3. Y45A—knockout of IL-2/IL-2R α interaction    -   4. L72G—knockout of IL-2/IL-2R α interaction    -   5. C125A—previously described mutation to avoid        disulfide-bridged IL-2 dimers

A mutant IL-2 polypeptide comprising all of mutations 1-4 is denotedherein as IL-2 quadruple mutant (qm). It may further comprise mutation 5(see SEQ ID NO: 19).

In addition to the three mutations F42A, Y45A and L72G designed tointerfere with the binding to CD25, the T3A mutation was chosen toeliminate the O-glycosylation site and obtain a protein product withhigher homogeneity and purity when the IL-2 qm polypeptide orimmunoconjugate is expressed in eukaryotic cells such as CHO or HEK293cells.

For purification purposes a His6 tag was introduced at the C-terminuslinked via a VD sequence. For comparison a non-mutated analogous versionof IL-2 was generated that only contained the C145A mutation to avoidundesired inter-molecular disulfide bridges (SEQ ID NO: 3). Therespective molecular weights without signal sequence were 16423 D fornaked IL-2 and 16169 D for the naked IL-2 qm. The wild-type andquadruple mutant IL-2 with His tag were transfected in HEK EBNA cells inserum-free medium (F17 medium) The filtered supernatant was bufferexchanged over a cross-flow, before loading it on a NiNTA SuperflowCartridge (5 ml, Qiagen). The column was washed with wash buffer 20 mMsodium phosphate, 0.5 M sodium chloride pH 7.4 and eluted with elutionbuffer. 20 mM sodium phosphate, 0.5 M sodium chloride 0.5 M imidazole pH7.4. After loading the column was washed with 8 column volumes (CV) washbuffer, 10 CV 5% elution buffer (corresponds to 25 mM imidazole), theneluted with a gradient to 0.5 M imidazole. The pooled eluate waspolished by size exclusion chromatography on a HiLoad 16/60 Superdex75(GE Healthcare) column in 2 mM MOPS, 150 mM sodium chloride, 0.02%sodium azide pH 7.3. FIG. 2 shows the chromatogram of the His tagpurification for the wild-type naked IL-2. Pool 1 was made fromfractions 78-85, pool 2 from fractions 86-111. FIG. 3 shows thechromatogram of the size exclusion chromatography for the wild-typeIL-2, for each pool the fractions 12 to 14 were pooled. FIG. 4 shows theanalytical size exclusion chromatography for wild-type IL-2 asdetermined on a Superdex 75, 10/300 GL (GE Healthcare) column in 2 mMMOPS, 150 mM sodium chloride, 0.02% sodium azide pH 7.3. Pool 1 and 2contained 2 proteins of ca. 22 and 20 kDa. Pool 1 had more of the largeprotein, and pool 2 had more of the small protein, putatively thisdifference is due to differences in O-glycosylation. Yields were ca. 0.5mg/L supernatant for pool 1 and ca. 1.6 mg/L supernatant for pool 2.FIG. shows the chromatogram of the His tag purification for thequadruple mutant IL-2. Pool 1 was made from fractions 59-91, pool 2 fromfractions 92-111. FIG. 6 shows the chromatogram of the size exclusionchromatography for the quadruple mutant IL-2, here only pool 2 fractions12 to 14 were kept. FIG. 7 shows the analytical size exclusionchromatography for the quadruple mutant IL-2 as determined on a Superdex75, 10/300 GL (GE Healthcare) column in 2 mM MOPS, 150 mM sodiumchloride, 0.02% sodium azide pH 7.3. The preparation for the nakedquadruple mutant IL-2 contained only one protein of 20 kD. This proteinhas the O-glycosylation site knocked out. Aliquots of the naked IL-2wild-type and quadruple mutant were stored frozen at −80° C. Yields wereca 0.9 mg/L supernatant.

A second batch of His-tagged quadruple mutant IL-2 was purified asdescribed above by immobilized metal ion affinity chromatography (IMAC)and followed by size exclusion chromatography-(SEC). The buffers usedfor IMAC were 50 mM Tris, 20 mM imidazole, 0.5M NaCl pH 8 for columnequilibration and washing, and 50 mM Tris, 0.5 M imidazole, 0.5 M NaClpH 8 for elution. The buffer used for SEC and final formulation bufferwas 20 mM histidine, 140 mM NaCl pH 6. FIG. 43 shows the result of thatpurification. The yield was 2.3 ml/L supernatant.

Subsequently, affinity for the IL-2R βγ heterodimer and the IL-2Rα-subunit were determined by surface plasmon resonance (SPR). Briefly,the ligand—either human IL-2R α-subunit (Fc2) or human IL2-R β knob γhole heterodimer (Fc3)—was immobilized on a CM5 chip. Subsequently,naked wild-type (pool 1 and 2) or quadruple mutant IL-2, and Proleukin(Novartis/Chiron) were applied to the chip as analytes at 25° C. inHBS-EP buffer in concentrations ranging from 300 nM down to 1.2 nM (1:3dil.). Flow rate was 30 l/min and the following conditions were appliedfor association: 180s, dissociation: 300 s, and regeneration: 2×30 s 3MMgCl₂ for IL2-R β knob γ hole heterodimer, 10 s 50 mM NaOH for IL-2Rα-subunit. 1:1 binding was applied for fitting (1:1 binding RI≠0,Rmax=local for IL-2R βγ, apparent K_(D), 1:1 binding RI=0, Rmax=localfor IL-2R α). Table 3 shows the respective K_(D) values for binding ofhuman wild-type and quadruple mutant IL-2 as well as of Proleukin toIL-2R βγ and IL-2R α-subunit.

TABLE 3 Affinity of mutant IL-2 polypeptides to the intermediateaffinity IL-2R and the IL-2R α-subunit. K_(D) in nM Hu IL-2R βγ Hu IL-2Rα Hu IL-2R α T = 25° C. (kinetic) (kinetic) (steady state) Naked IL-2wt, pool 1 5.6 17.4 30.3 5 16.6 23.9 Naked IL-2 wt, pool 2 2.8 10.6 19.71.8 10 17.6 Naked IL-2 qm 2.7 no binding no binding 2 Proleukin 2.4 7.519 2.8 12.5 17.8

The data show that the naked IL-2 quadruple mutant shows the desiredbehaviour and has lost binding for the IL-2R α-subunit whereas bindingto IL-2R βγ is retained and comparable to the respective wild type IL-2construct and Proleukin. Differences between pools 1 and 2 of thewild-type IL-2 can probably be attributed to differences inO-glycosylation. This variability and heterogeneity has been overcome inthe IL-2 quadruple mutant by introduction of the T23A mutation.

Example 3

The three mutations F42A, Y45A and L72G and the mutation T3A wereintroduced in the Fab-IL-2-Fab format (FIG. 1A) using the anti-FAPantibody 4G8 as model targeting domain either as single mutants: 1) 4G8IL-2 T3A, 2) 4G8 IL-2 F42A, 3) 4G8 IL-2 Y45A, 4) 4G8 IL-2 L72G, or theywere combined in Fab-IL-2 mt-Fab constructs as: 5) triple mutantF42A/Y45A/L72G, or as: 6) quadruple mutant T3A/F42A/Y45A/L72G toinactivate the O-glycosylation site as well. The 4G8-based Fab-IL-2wt-Fab served for comparison. All constructs contained the C145Amutation to avoid disulfide-bridged IL-2 dimers. The different Fab-IL2-Fab constructs were expressed in HEK 293 cells and purified asdescribed above via protein A and size exclusion chromatography asspecified above. Subsequently, the affinity of the selected IL 2variants for the human and murine IL 2R βγ heterodimer and for the humanand murine IL-2R α-subunit was determined by surface plasmon resonance(SPR) (Biacore) using recombinant IL-2R βγ heterodimer and monomericIL-2R α-subunit under the following conditions: The IL-2R α-subunit wasimmobilized in two densities and the flow cell with higherimmobilization was used for the mutants that have lost CD25 binding. Thefollowing conditions were used: chemical immobilization: human IL-2R byheterodimer 1675 RU; mouse IL-2R βγ heterodimer 5094 RU; human IL-2Rα-subunit 1019 RU; human IL-2R α-subunit 385 RU, murine IL-2R α-subunit1182 RU; murine IL-2R α-subunit 378 RU, temperature: 25° C., analytes:4G8 Fab-IL 2 variants-Fab constructs 3.1 nM to 200 nM, flow 40 μl/min,association: 180 s, dissociation: 180 s, regeneration: 10 mM glycine pH1.5, 60 s, 40 μl/min. Fitting: two state reaction model (conformationalchange), RI=0 Rmax=local. Results of the kinetic analysis are given inTable 4.

TABLE 4 Affinity of FAP-targeted immunoconjugates comprising mutant IL-2polypeptides to the intermediate affinity IL-2R and the IL-2R α-subunit(K_(D)). Construct Hu Fab-IL-2-Fab Hu IL-2R βγ IL-2R α Mu IL-2R βγ MuIL-2R α 4G8 IL-2 wt 3.8 nM  4.5 nM 45.6 nM  29 nM 4G8 IL-2 T3A 1.6 nM 4.9 nM 15.6 nM  15 nM 4G8 IL-2 F42A 4.7 nM  149 nM   57 nM 363 nM 4G8IL-2 Y45A 3.9 nM 22.5 nM 41.8 nM 369 nM 4G8 IL-2 L72G ND 45.3 nM ND ND4G8 IL-2 triple 5.6 nM no binding 68.8 nM ND mutant F42A/ Y45A/L72G 4G8IL-2 5.2 nM no binding 56.2 nM no binding quadruple mutant T3A/F42A/Y45A/L72G

Simultaneous binding to the IL 2R βγ heterodimer and FAP was shown bySPR. Briefly, the human IL 2R βγ knob-into-hole construct wasimmobilized on a CM5 chip chemically and 10 nM Fab-IL-2-Fib constructswere captured for 90 s. Human FAP served as analyte at concentrations of200 nM down to 0.2 nM. Conditions were: temperature: 25° C., buffer:HBS-EP, flow: 30 μl/min, association: 90 s, dissociation: 120 s.Regeneration was done for 60 s with 10 mM glycine pH 2. Fitting wasperformed with a model for 1:1 binding, RI≠0, Rmax-global. The SPRbridging assay showed that the Fab-IL-2-Fab constructs, both aswild-type and as quadruple mutant, as well as based on the affinitymatured FAP binder 28H1 or the parental 3F2 or 4G8 antibodies, was-ableto bind at a concentration of 10 nM simultaneously to the IL 2R βγheterodimer immobilized on the chip as well as to human FAP used asanalyte (FIG. 8). The determined affinities are shown in Table 5.

TABLE 5 Affinity of FAP-targeted immunoconjugates, comprising mutantIL-2 polypeptides and bound to the intermediate affinity IL-2R, to FAP(K_(D)). Construct Fab-IL-2-Fab K_(D) 4G8 Fab-IL-2 wt-Fab 5.0 nM 4G8Fab-IL-2 qm-Fab 5.6 nM 29B11 Fab-IL-2 wt-Fab 0.32 nM  29B11 Fab-IL-2qm-Fab 0.89 nM  3F2 Fab-IL-2 wt-Fab 1.2 nM

Taken together the SPR data showed that i) the T3A mutation does notinfluence binding to CD25, ii) the three mutations F42A, Y45A and L72Gdo not influence the affinity for the IL 2R βγ heterodimer while theyreduce the affinity for CD25 in this order: wt=T3A>Y45A (ca. 5×lower)>L72G (ca. 10× lower)>F42A (ca. 33× lower); iii) the combinationof the three mutations F42A, Y45A and L72G with or without theO-glycosylation site mutant T3A results in a complete loss of CD25binding as determined under SPR conditions, iv) although affinity ofhuman IL-2 for murine IL-2R βγ heterodimer and IL-2R α-subunit isreduced approximately by a factor of 10 compared to human IL-2 receptorsthe selected mutations do not influence affinity for the murine IL-2R βγheterodimer, but abolish binding to murine IL-2R α-subunit accordingly.This indicates that the mouse represents a valid model for the study ofpharmacological and toxicological effects of IL-2 mutants, althoughoverall IL-2 exhibits less toxicity in rodents than in humans.

Apart from the loss of O-glycosylation one additional advantage of thecombination of the four mutations T3A, F42A, Y45A, L72G is a lowersurface hydrophobicity of the IL-2 quadruple mutant due to the exchangeof surface exposed hydrophobic residues such as phenylalanine, tyrosineor leucine by alanine. An analysis of the aggregation temperature bydynamic light scattering showed that the aggregation temperature for theFAP-targeted Fab-IL-2-Fab immunoconjugates comprising wild-type orquadruple mutant IL-2 were in the same range: ca. 57-58° C. for the 3F2parental Fab-IL-2-Fab and for the affinity matured 29B11 3F2-derivative;and in the range of 62-63° C. for the 4G8 parental Fab-IL-2-Fab and theaffinity matured 28H1, 4B9 and 14B3 4G8-derivatives, indicating that thecombination of the four mutations had no negative impact on proteinstability. In support of the favorable properties of the selected IL-2quadruple mutant, transient expression yields indicated that thequadruple mutant in the Fab-IL-2 qm-Fab format may even result in higherexpression yields than those observed for the respective Fab-IL-2 wt-Fabconstructs. Finally, pharmacokinetic analysis shows that both 4G8-basedFab-IL-2 qm-Fab and Fab-IL-2 wt-Fab have comparable PK properties (seeexample 9 below). Based on these data and the cellular data described inexample 4 below the quadruple mutant T3A, F42A, Y45A, L72G was selectedas ideal combination of mutations to abolish CD25 binding of IL-2 in thetargeted Fab-IL-2-Fab immunoconjugate.

Example 4

The 4G8-based FAP-targeted Fab-IL 2-Fab immunoconjugates, comprisingwild-type IL-2 or the single mutants 4G8 IL-2 T3A, 4G8 IL-2 F42A, 4G8IL-2 Y45A, 4G8 IL-2 L72G or the respective triple (F42A/Y45A/L72G) orquadruple mutant (T3A/F42A/Y45A/L72G) IL-2, were subsequently tested incellular assays in comparison to Proleukin as described above.

IL-2 induced IFN-γ release was measured following incubation of the NKcell line NK92 with the constructs (FIG. 9). NK92 cells express CD25 ontheir surface. The results show that the Fab-IL-2-Fab immunoconjugatecomprising wild-type IL-2 was less potent in inducing IFN-γ release thanProleukin as could be expected from the ca. 10-fold lower affinity ofthe Fab-IL-2 wt-Fab for the IL-2R βγ heterodimer. The introduction ofsingle mutations interfering with CD25 binding as well as thecombination of the three mutations interfering with CD25 binding in theIL-2 triple mutant resulted in Fab-IL-2-Fab constructs that werecomparable to the wild-type IL-2 construct in terms of potency andabsolute induction of IFN-γ release within the error of the method.

TABLE 6 Induction of IFN-γ release from NK cells by Fab-IL-2-Fabimmunoconjugates comprising mutant IL-2 polypeptides. Construct EC₅₀[nM] Proleukin 4.1 4G8 Fab-IL 2 wt-Fab 23.0 4G8Fab-IL-2 (T3A)-Fab 16.24G8 Fab-IL-2 (F42A)-Fab 15.4 4G8 Fab-IL-2(Y45A)-Fab 20.9 4G8 Fab-IL-2(L72G)-Fab 16.3 4G8 Fab-IL-2 (triple mutant 42/45/72)-Fab 24.4

Subsequently, induction of proliferation of isolated human NK cells byFab-IL-2-Fab immunoconjugates was assessed in a proliferation assay(Cell Titer Glo, Promega) (FIG. 10). In contrast to NK92 cells, freshlyisolated NK cells do not express CD25 (or only very low amounts). Theresults show that the Fab-IL-2-Fab immunoconjugate comprising wild-typeIL-2 was ca. 10-fold less potent in inducing NK cell proliferation thanProleukin, as could be expected from the ca. 10-fold lower affinity ofthe Fab-IL-2 wt-Fab immunoconjugate for the IL-2R βγ heterodimer. Theintroduction of single mutations interfering with CD25 binding as wellas the combination of the three mutations interfering with CD25 in theIL-2 triple mutant resulted in Fab-IL-2-Fab constructs that werecomparable to the wild-type IL-2 construct in terms of potency andabsolute induction of proliferation; there was only a very small shiftin potency observed for the Fab-IL-2-Fab triple mutant. In a secondexperiment the induction of proliferation of PHA-activated T cells wasassessed following incubation with different amounts of Proleukin andFab-IL-2-Fab immunoconjugates (FIG. 11). As activated T cells expressCD25, a clear reduction in T cell proliferation could be observed uponincubation with the immunoconjugates comprising IL-2 single mutantsF42A, L72G or Y45A; with F42A showing the strongest reduction followedby L72G and Y45A, whereas when using Fab-IL-2 wt-Fab or Fab-IL-2(T3A)-Fab the activation was almost retained compared to Proleukin.These data reflect the reduction in affinity for CD25 as determined bySPR (example above). The combination of the three mutations interferingwith CD25 binding in the IL-2 triple mutant resulted in animmunoconjugate that mediated significantly reduced induction of T cellproliferation in solution. In line with these findings we measured celldeath of T cells as determined by Annexin V/PI staining followingover-stimulation induced by a first stimulation for 16 h with 1 μg/mlPHA, a second stimulation for 5 days with Proleukin or the respectiveFab-IL-2-Fab immunoconjugates, followed by a third stimulation with 1μg/ml PHA. In this setting we observed that activation induced celldeath (AICD) in over-stimulated T cells was strongly reduced with theFab-IL-2-Fab immunoconjugates comprising the IL-2 single mutants F42A,L72G and Y45A interfering with CD25 binding with F42A and L720 showingthe strongest reduction, which was similar to the reduction achieved bythe combination of the three mutations in the immunoconjugate comprisingthe IL-2 triple mutant (FIG. 12). In a last set of experiments westudied the effects of the Fab-IL-2 qm-Fab on the induction of STAT5phosphorylation compared to Fab-IL-2 wt-Fab and Proleukin on human NKcells, CD4⁺ T cells, CD8⁺ T cells and T_(reg) cells from human PBMCs(FIG. 13). For NK cells and CD8⁺ T cells that show no or very low CD25expression (meaning that IL-2R signaling is mediated via the IL-2R βγheterodimer) the results show that the Fab-IL-2-Fab format comprisingwildtype IL-2 was ca. 10-fold less potent in inducing STAT5phosphorylation than Proleukin, and that the Fab-IL-2 qm-Fab wascomparable to the Fab-IL-2 wt-Fab construct. On CD4⁺ T cells, that showa rapid up-regulation of CD25 upon stimulation, the Fab-IL-2 qm-Fab wasless potent then the Fab-IL-2 wt-Fab immunoconjugate, but still showedcomparable induction of IL-2R signaling at saturating concentrations.This is in contrast to T_(reg) cells where the potency of the Fab-IL-2qm-Fab was significantly reduced compared to the Fab-IL-2 wt-Fabimmunoconjugate due to the high CD25 expression on T_(reg) cells and thesubsequent high binding affinity of the Fab-IL-2 wt-Fab immunoconjugateto CD25 on T_(reg) cells. As a consequence of the abolishment of CD25binding in the Fab-IL-2 qm-Fab immunoconjugate, IL-2 signaling inT_(reg) cells is only activated via the IL-2R βγ heterodimer atconcentrations where IL-2R signaling is activated on CD25-negativeeffector cells through the IL-2R βγ heterodimer. Taken together the IL-2quadruple mutant described here is able to activate IL-2R signalingthrough the IL-2R βγ heterodimer, but does neither result in AICD nor ina preferential stimulation of T_(reg) cells over other effector cells.

Example 5

Based on the data described in examples 2 and 3 affinity maturedFAP-targeted Fab-IL-2 qm-Fab immunoconjugates based on clones 28H1 or29B11 were generated and purified as described above in the generalmethods section. In more detail, the FAP-targeted 28H1 targeted Fab-IL-2qm-Fab was purified by one affinity step (protein G) followed by sizeexclusion chromatography (Superdex 200). Column equilibration wasperformed in PBS and supernatant from a stable CHO pool (CDCHO medium)was loaded onto a protein G column (GE Healthcare), the column waswashed with PBS and samples were subsequently eluted with 2.5 mM HCl andfractions were immediately neutralized with 10×PBS. Size exclusionchromatography was performed in the final formulation buffer 25 mMsodium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7 on aSuperdex 200 column. FIG. 14 shows the elution profiles from thepurification and the results from the analytical characterization of theproduct by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel 4-20%, Invitrogen,MOPS running buffer, reduced and non-reduced). Given the low bindingcapacity of the 28H1 Fab fragment to protein 3 and protein A additionalcapture steps may result in higher yields.

FAP-targeted 408, 3F2 and 29B11 Fab-IL-2 qm-Fab and MCSP-targeted MHLG1KV9 Fab-IL-2 qm-Fab immunoconjugates were purified by one affinity step(protein A) followed by size exclusion chromatography (Superdex 200).Column equilibration was performed in 20 mM sodium phosphate, 20 mMsodium citrate pH 7.5 and supernatant was loaded onto the protein Acolumn. A first wash was performed in 20 mM sodium phosphate, 20 mMsodium citrate, pH 7.5 followed by a second wash: 13.3 mM sodiumphosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. TheFab-IL-2 qm-Fab immunoconjugates were eluted in 20 mM sodium citrate,100 mM sodium chloride, 100 mM glycine pH 3. Size exclusionchromatography was performed in the final formulation buffer: 25 mMpotassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7. FIG.15 shows the elution profiles from the purification and the results fromthe analytical characterization of the product by SDS-PAGE (NuPAGE NovexBis-Tris Mini Gel 4-20%, Invitrogen, MOPS running buffer, reduced andnon-reduced) for the 4G8 Fab-IL-2 qm-Fab and FIG. 16 for the MHLG1 KV9Fab-IL-2 qm-Fab immunoconjugate.

FAP-targeted IgG-IL-2 qm fusion proteins based on the FAP-antibodies4G8, 4B9 and 28H1, and a control DP47GS non-targeted IgG-IL-2 qm fusionprotein were generated as described above in the general methodssection. FIGS. 46 and 47 show the respective chromatograms and elutionprofiles of the purification (A, B) as well as the analytical SDS-PAGEand size exclusion chromatographies of the final purified constructs (C,D) for the 4G8- and 28H1-based constructs. Transient expression yieldswere 42 mg/L for the 4G8-based and 20 mg/L for the 28H1-based IgG-IL-2qm immunoconjugate.

The FAP binding activity of the IgG-IL-2 qm immunoconjugates based on4G8 and 28H1 anti-FAP antibodies were determined by surface plasmonresonance (SPR) on a Biacore machine in comparison to the correspondingunmodified IgG antibodies. Briefly, an anti-His antibody (Penta-His,Qiagen 34660) was immobilized on CM5 chips to capture 10 nM His-taggedhuman FAP (20 s). Temperature was 25° C. and HBS-EP was used as buffer.Analyte concentration was 50 nM down to 0.05 nM at a flow rate of 50μl/min (association: 300 s, dissociation: 900 s, regeneration: 60 s,with 10 mM glycine pH 2). Fitting was performed based on a 1:1 bindingmodel, RI=0, Rmax=local (because of capture format). Table 7 gives theestimated apparent bivalent affinities (pM avidity) as determined by SPRfitted with 1:1 binding RI=0, Rmax-local.

TABLE 7 K_(D) [pM] Hu FAP 4G8 IgG-IL-2 qm 100 4G8 IgG 50 28H1 IgG-IL-2qm 175 28H1 IgG 200

The data show that within the error of the method affinity for human FAPis retained for the 28H1-based immunoconjugate or only slightlydecreased for the 4G8-based immunoconjugate as compared to thecorresponding unmodified antibodies.

Example 6

The affinity of the FAP-targeted, affinity matured 28H1 and 29B11-basedFab-IL-2-Fab immunoconjugates, each comprising wild-type or quadruplemutant IL-2, and of the 3F2-based Fab-IL-2 wt-Fab were determined bysurface plasmon resonance (SPR) for the human, murine and cynomolgusIL-2R βγ heterodimer using recombinant IL-2R βγ heterodimer under thefollowing conditions: ligand: human, murine and cynomolgus IL-2R β knobγ hole heterodimer immobilized on CM5 chip, analyte: 28H1 or 29B11Fab-IL-2-Fab (comprising wild-type or quadruple mutant IL-2), 3F2Fab-IL-2-Fab (comprising wild-type IL-2), temperature: 25° C. or 37° C.,buffer HBS-EP, analyte concentration: 200 nM down to 2.5 nM, flow: 30μl/min, association: 300 s, dissociation: 300 s, regeneration: 60 s 3MMgCl₂, fitting: 1:1 binding. RI≠0, Rmax=global. The affinity of theFAP-targeted affinity matured 28H1 and 29B11-based Fab-IL-2-Fabimmunoconjugate, each containing wildtype or quadruple mutant IL-2, andof the 3F2-based Fab-IL-2 wt-Fab were determined by surface plasmonresonance (SPR) for the human, murine and cynomolgus IL-2R α-subunitusing recombinant monomeric IL-2R α-subunit under the followingconditions: ligand: human, murine and cynomolgus IL-2R α-subunitimmobilized on a CM5 chip, analyte: 28H1 or 29B11 Fab-IL-2-Fab(comprising wild-type or mutant IL-2), 3F2 Fab-IL-2-Fab (comprisingwild-type IL-2), temperature: 25° C. or 37° C., buffer: HBS-EP, analyteconcentration 25 nM down to 0.3 nM, flow: 30 μl/min, association: 120 s,dissociation: 600 s, regeneration: none, fitting: 1:1 binding, RI=0,Rmax=global.

Results of the kinetic analysis with the IL-2R βγ heterodimer are givenin Table 8.

TABLE 8 Binding of Fab-IL-2-Fab immunoconjugates comprising affinitymatured Fab and mutant IL-2 to IL-2R βγ heterodimers. Hu IL-2R Hu IL-2RCyno IL-2R Cyno IL-2R Mu IL-2R Mu IL-2R K_(D) in nM βγ (25° C.) βγ (37°C.) βγ (25° C.) βγ (37° C.) βγ (25° C.) βγ (37° C.) 28H1 Fab-IL- 9.7 1911.5 29.2 112 186 2 wt-Fab 9 22 11.6 30.4 79 219 28H1 Fab-IL- 7.5 14.38.9 21.3 66 142 2 qm-Fab 6.9 14.7 8.4 21.2 54 106 29B11 Fab- 6.5 9.5 6.914 93 71 IL-2 wt-Fab 5.7 12.4 6.7 19 74 74 29B11 Fab-IL- 12 13.1 7.816.7 60 44 2 qm-Fab 7.4 13 8.4 18.1 63 42 3F2 Fab-IL- 5 ND 6.4 ND 40 ND2 wt-Fab 4.8 6.1 40

Whereas the affinity of human IL-2 to the human IL 2R βγ heterodimer isdescribed to be around 1 nM, the Fab-IL-2-Fab immunoconjugates(comprising wild-type or quadruple mutant IL-2) both have a reducedaffinity between 6 and 10 nM, and as shown for the naked IL-2 above theaffinity to the murine IL-2R is around 10 times weaker than for thehuman and cynomolgus IL-2R.

Results of the kinetic analysis with the IL-2R α-subunit are given inTable 9. Under the chosen conditions there is no binding detectable ofthe immunoconjugates comprising the IL-2 quadruple mutant to the human,murine or cyno IL-2R α-subunit.

TABLE 9 Binding of Fab-IL-2-Fab immunoconjugates comprising affinitymatured Fab and mutant IL-2 to IL-2R α-subunits. Hu IL-2R Hu IL-2R CynoIL-2R Cyno IL-2R Mu IL-2R Mu IL-2R K_(D) in nM α (25° C.) α (37° C.) α(25° C.) α (37° C.) α (25° C.) α (37° C.) 28H1 Fab-IL- 16 28.8 16 36.543.3 67.5 2 wt-Fab 16.2 28.2 16.2 35.6 44   61.1 28H1 Fab-IL- no no nono no no 2 qm-Fab binding binding binding binding binding binding 29B11Fab- 5  7.6 4.8  7.3 11.4 13.3 IL-2 wt-Fab 4.6  7.7 4.3  7.4  9.6 13.829B11 Fab- no no no no no no IL-2 qm-Fab binding binding binding bindingbinding binding 3F2 Fab-IL- 5.7 ND 5 ND 12.3 ND 2 wt-Fab 6.1 5.4 12.1

The affinity of the MCSP-targeted MHLG1-KV9 Fab-IL-2-Fabimmunoconjugates, comprising the wild-type or quadruple mutant IL-2,were determined by surface plasmon resonance (SPR) for the human IL-2Rβγ heterodimer using recombinant IL-2R βγ heterodimer under thefollowing conditions: human IL-2R β knob γ hole heterodimer wasimmobilized on a CM5 chip (1600 RU). MHLG1-KV9 Fab-IL-2 wt-Fab andFab-IL-2 qm-Fab were used as analyte at 25° C. in HBS-P buffer. Analyteconcentration was 300 nM down to 0.4 nM (1:3 dil.) for IL-2R βγ at aflow of 30 p/min (association time 180 s, dissociation time 300 s).Regeneration was done for 2×30 s with 3M MgCl₂ for IL-2R βγ. Data werefitted using a 1:1 binding, RI≠0, Rmax=local for IL-2R βγ.

The affinity of the MCSP-targeted MHLG1-KV9 Fab-IL-2-Fabimmunoconjugates, comprising the wild-type or quadruple mutant IL-2,were determined by surface plasmon resonance (SPR) for the human IL-2Rα-subunit using recombinant monomeric IL-2R α-subunit under thefollowing conditions: human IL-2R α-subunit was immobilized on a CM5chip (190 RU). MHLG1-KV9 Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab were usedas analyte at 25° C. in HBS-P buffer. Analyte concentration was 33.3 nMdown to 0.4 nM (1:3 dil.) for IL-2R α at a flow of 30 μl/min(association time 180 s, dissociation time 300 s). Regeneration was donefor 10 s with 50 mM NaOH for IL-2R α. Data were fitted using a 1:1binding, RI=0, Rmax-global for IL-2R α.

Results of the kinetic analysis with the IL-2R βγ heterodimer are givenin Table 10.

TABLE 10 K_(D) in nM Hu IL 2R βγ Hu IL 2R α Hu IL 2R α T = 25° C.(kinetic) (kinetic) (steady state) MHLG1-KV9 Fab-IL-2 8.6 8.8 6.8 wt-Fab9.8 10.1 10.9 MHLG1-KV9 Fab-IL 2 7.3 No binding No binding qm-Fab 10.7

The data confirm that the MCSP-targeted MHLG1-KV9 Fab-IL-2 qm-Fabimmunoconjugate has retained affinity for the IL-2R receptor, whereasbinding affinity to CD25 is abolished compared to the immunoconjugatecomprising wild-type IL-2.

Subsequently, the affinity of the 4G8- and 28H1-based IgG-IL-2 qmimmunoconjugates to the IL-2R βγ heterodimer and the IL-2R α-subunitwere determined by surface plasmon resonance (SPR) in direct comparisonto the Fab-IL-2 qm-Fab immunoconjugate format. Briefly, theligands—either the human IL-2R α-subunit or the human IL-2R βγheterodimer—were immobilized on a CM5 chip. Subsequently, the 4G8- and28H1-based IgG-IL-2 qm immunoconjugates or the 4G8- and 28H1-basedFab-IL-2 qm-Fab immunoconjugates were applied to the chip as analytes at25° C. in HBS-EP buffer in concentrations ranging from 300 nM down to1.2 nM (1:3 dil.). Flow rate was 30 μl/min and the following conditionswere applied for association: 180s, dissociation: 300 s, andregeneration: 2×30 s with 3 M MgCl₂ for IL-2R βγ heterodimer, 10 s with50 mM NaOH for IL-2R α-subunit. 1:1 binding was applied for fitting (1:1binding RI≠0, Rmax=local for IL-2R βγ, apparent K_(D), 1:1 binding RI=0,Rmax-local for IL-2R α). The respective K_(D) values are given in Table11.

TABLE 11 Apparent K_(D) [nM] Hu IL-2R βγ Hu IL-2R α 4G8 IgG-IL-2 qm 5.9No binding 4G8 Fab-IL-2 qm-Fab 10.4 No binding 28H1 IgG-IL-2 qm 6.2 Nobinding 28H1 Fab-IL-2 qm-Fab 11.4 No binding

The data show that the 4G8- and 28H1-based IgG-IL-2 qm immunoconjugatesbind with at least as good affinity as the Fab-IL-2 qm-Fabimmunoconjugates to the IL-2βγ heterodimer, whereas they do not bind tothe IL-2R α-subunit due to the introduction of the mutations interferingwith CD25 binding. Compared to the corresponding Fab-IL-2 qm-Fabimmunoconjugates the affinity of the IgG-IL-2 qm fusion proteins appearsto be slightly enhanced within the error of the method.

Example 7

In a first set of experiments we confirmed that the FAP-targetedFab-IL-2-Fab immunoconjugates comprising either wild-type or mutant IL-2were able to bind to human FAP-expressing HEK.293-FAP cells by FACS(FIG. 17) and that the IL-2 quadruple mutation did not impact binding toFAP-expressing cells (FIG. 18).

TABLE 12 Binding of Fab-IL-2-Fab immunoconjugates to FAP-expressing HEKcells. EC₅₀ values nM 28H1 Fab-IL-2-Fab 0.64 28H1 Fab-IL-2 qm-Fab 0.7029B11 Fab-IL-2-Fab 0.66 29B11 Fab-IL-2 qm-Fab 0.85 4G8 Fab-IL-2-Fab 0.65

In particular, these binding experiments showed that the affinitymatured FAP binders 28H1, 29B11, 14B3 and 4B9 as Fab-IL-2 qm-Fab showedsuperior absolute binding to the HEK 293-FAP target cells compared tothe Fab-IL-2-Fab immunoconjugates based on the parental FAP binders 3F2(29B11, 14B3, 4B9) and 4G8 (28H1) (FIG. 17), while retaining highspecificity and no binding to HEK 293 cells transfected with DPPIV, aclose homologue of FAP, or HEK 293 mock-transfected cells. Forcomparison the mouse anti-human CD26-PE DPPIV antibody clone M-A261 (BDBiosciences, #555437) was used as a positive control (FIG. 19). Analysisof the internalization properties showed that the binding ofFab-IL-2-Fab immunoconjugates do not result in the induction of FAPinternalization (FIG. 20).

In a further experiment, binding of FAP-targeted 4G8-based IgG-IL-2 qmand Fab-IL-2 qm-Fab immunoconjugates to human FAP expressed on stablytransfected HEK293 cells was measured by FACS. The results are shown inFIG. 48. The data show that the IgG-IL-2 qm immunoconjugate binds toFAP-expressing cells with an EC50 value of 0.9 nM, comparable to that ofthe corresponding 4G8-based Fab-IL-2 qm-Fab construct (0.7 nM).

The affinity matured anti-FAP Fab-IL-2-Fab immunoconjugates comprisingwildtype IL-2 or the quadruple mutant were subsequently tested incellular assays in comparison to Proleukin as described in the examplesabove.

IL-2 induced IFN-γ release was measured in the supernatant by ELISAfollowing incubation of the NK-cell line NK92 with theseimmunoconjugates (FIG. 21) for 24 h. NK92 cells express CD25 on theirsurface. The results show that the Fab-IL-2-Fab immunoconjugatecomprising wild-type IL-2 was less potent in inducing IFN-γ release thanProleukin as could be expected from the ca. 10-fold lower affinity ofthe Fab-IL-2 wt-Fab immunoconjugate for the IL-2R βγ heterodimer. TheFab-IL-2 qm-Fab immunoconjugates were quite comparable to the respectivewild-type construct for a selected clone in terms of potency andabsolute induction of IFN-γ release despite the fact that NK92 cellsexpress some CD25. It could, however, be observed that the 29B 11Fab-IL-2 qm-Fab induced less cytokine release compared to the 29B11Fab-IL-2 wt-Fab as well as the 28H1 and 4G8 constructs, for which therewas only a small shift in potency observed for Fab-IL-2 qm-Fab overFab-IL-2 wt-Fab.

In addition, the MCSP-targeted MHLG1-KV9-based Fab-IL-2 qm-Fabimmunoconjugate was compared to the 28H1 and 29B11 based Fab-IL-2 qm-Fabimmunoconjugates in the IFN-γ release assay on NK92 cells. FIG. 22 showsthat the MCSP-targeted MHLG1-KV9-based Fab-IL-2 qm-Fab is quitecomparable in inducing IFN-γ release to the FAP-targeted Fab-IL-2 qm-Fabimmunoconjugates.

Subsequently, induction of proliferation of NK92 cells by IL-2 over aperiod of 3 days was assessed in a proliferation assay by ATPmeasurement using CellTiter Glo (Promega) (FIG. 23). Given that NK92cells express low amounts of CD25, a difference between Fab-IL-2-Fabimmunoconjugates comprising wild-type IL-2 and immunoconjugatescomprising quadruple mutant IL-2 could be detected in the proliferationassay, however, under saturating conditions both achieved similarabsolute induction of proliferation.

In a further experiment we studied the effects of the 28H1 affinitymatured FAP-directed Fab-IL-2 qm-Fab immunoconjugate on induction ofSTAT5 phosphorylation compared to 28H1 Fab-IL-2 wt-Fab and Proleukin onhuman NK cells, CD4⁺ T cells, CD8⁺ T cells and T_(reg) cells from humanPBMCs. (FIG. 24). For NK cells and CD8⁺ T cells, that show no or verylow CD25 expression (meaning that IL-2R signaling is mediated via theIL-2R βγ heterodimer), the results showed that the Fab-IL-2-Fabimmunoconjugate comprising wild-type IL-2 was ca. >10-fold less potentin inducing IFN-γ release than Proleukin, and that the Fab-IL-2 qm-Fabimmunoconjugate was only very slightly less potent than the Fab-IL-2wt-Fab construct. On CD4⁺ T cells that show a rapid up-regulation ofCD25 upon stimulation, the Fab-IL-2 qm-Fab was significantly less potentthan the Fab-IL-2 wt-Fab immunoconjugate, but still showed comparableinduction of IL-2R signaling at saturating concentrations. This is incontrast to T_(reg) cells, where the potency of the Fab-IL-2 qm-Fab wassignificantly reduced compared to the Fab-IL-2 wt-Fab construct due tothe high CD25 expression on T_(reg) cells and the subsequent highbinding affinity of the Fab-IL-2 wt-Fab construct to CD25 on T_(reg)cells. As a consequence of the abolishment of CD25 binding in theFab-IL-2 qm-Fab immunoconjugate, IL-2 signaling in T, cells is onlyactivated via the IL-2R βγ heterodimer at concentrations where IL-2Rsignaling is activated on CD25 negative effector cells through the IL-2Rβγ heterodimer. The respective pM EC50 values are given in Table 13.

TABLE 13 Induction of IFN-γ release from NK cells by 28H1 FAP-targetedFab-IL-2-Fab immunoconjugates comprising mutant IL-2 polypeptides. NKCD8⁺ CD4⁺ T_(reg) EC₅₀ [pM] cells T cells T cells cells Proleukin 2221071 92 1 28H1 Fab-IL-2 wt-Fab 3319 14458 3626 15 28H1 Fab-IL-2 qm-Fab3474 20583 70712 19719

In another set of experiments, the biological activity of FAP-targeted4G8-based IgG-IL-2 qm and Fab-IL-2 qm-Fab immunoconjugates wasinvestigated in several cellular assays.

FAP-targeted 4G8-based IgG-IL-2 qm and 28H1-based Fab-IL-2 qm-Fabimmunoconjugates were studied for the induction of IFN-γ release by NK92cells as induced by activation of IL-2R βγ signaling. FIG. 49 shows thatthe FAP-targeted 4G8-based IgG-IL-2 qm immunoconjugate was equallyefficacious in inducing IFN-γ release as the affinity matured 28H1-basedFab-IL-2 qm-Fab immunoconjugate.

We also studied the effects of the FAP-targeted 4G8-based IgG-IL-2 qmimmunoconjugate on the induction of STAT5 phosphorylation compared tothe 28H1 based Fab-IL-2 wt-Fab and Fab-IL-2 qm-Fab immunoconjugates aswell as Proleukin on human NK cells, CD4⁺ T cells, CD8⁺ T cells andT_(reg) cells from human PBMCs. The results of these experiments areshown in FIG. 50. For NK cells and CD8⁺ T cells the 4G8-based IgG-IL-2qm immunoconjugate was <10-fold less potent in inducing STAT5phosphorylation than Proleukin, but slightly more potent than 28H1-basedFab-IL-2 wt-Fab and Fab-IL-2 qm-Fab immunoconjugates. On CD4⁺ T cellsthe 4G8-based IgG-IL-2 qm immunoconjugate was less potent than the 28H1Fab-IL-2 wt-Fab immunoconjugate, but slightly more potent than the 28H1Fab-IL-2 qm-Fab immunoconjugate, and still showed induction of IL-2Rsignaling at saturating concentrations comparable to Proleukin and 28H1Fab-IL-2 wt-Fab. This is in contrast to T_(reg) cells where the potencyof the 4G8-based IgG-IL-2 qm and 28H1 Fab-IL-2 qm-Fab immunoconjugateswas significantly reduced compared to the Fab-IL-2 wt-Fabimmunoconjugate.

Taken together the IL-2 quadruple mutant described here is able toactivate IL-2R signaling through the IL-2R βγ heterodimer similar towild-type IL-2, but does not result in a preferential stimulation ofT_(reg) cells over other effector cells.

Example 8

The anti-tumoral effects of FAP-targeted Fab-IL-2 qm-Fabimmunoconjugates were evaluated in vivo in comparison to FAP-targetedFab-IL-2 wt-Fab immunoconjugates in ACHN xenograft and LLC1 syngeneicmodels. All FAP-targeted Fab-IL-2-Fab immunoconjugates (comprisingwild-type or quadruple mutant IL-2) recognize murine FAP as well as themurine IL-2R. While the ACHN xenograft model in SCID-human FcγRIIItransgenic mice is strongly positive for FAP in IHC, it is animmunocompromised model and can only reflect immune effector mechanismsmediated by NK cells and/or macrophages/monocytes, but lacks T cellmediated immunity and thus cannot reflect AICD or effects mediatedthrough T_(reg) cells. The syngeneic LLC1 model in contrast in fullyimmunocompetent mice can reflect adaptive T cell mediated immuneeffector mechanisms as well, but shows fairly low expression of FAP inthe murine stroma. Each of these models thus partially reflects thesituation as encountered in human tumors.

ACHN Renal Cell Carcinoma Xenograft Model

The FAP-targeted 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fabimmunoconjugates were tested using the human renal cell adenocarcinomacell line ACHN, intra-renally injected into SCID-human FcγRIIItransgenic mice. ACHN cells were originally obtained from ATCC (AmericanType Culture Collection) and after expansion deposited in the Glycartinternal cell bank. ACHN cells were cultured in DMEM containing 10% FCS,at 37° C. in a water-saturated atmosphere at 5% CO₂. In vitro passage 18was used for intrarenal injection, at a viability of 98.4%. A smallincision (2 cm) was made at the right flank and peritoneal wall ofanesthetized SCID mice. Fifty μl cell suspension (1×10⁶ ACHN cells inAimV medium) was injected 2 mm subcapsularly in the kidney. Skin woundsand peritoneal wall were closed using clamps. Female SCID-FcγRIII mice(GLYCART-RCC), aged 8-9 weeks at the beginning of the experiment (bredat RCC, Switzerland) were maintained under specific-pathogen-freeconditions with daily cycles of 12 h light/12 h darkness according tocommitted guidelines (GV-Solas; Felasa; TierschG). The experimentalstudy protocol was reviewed and approved by local government (P2008016). After arrival, animals were maintained for one week to getaccustomed to new environment and for observation. Continuous healthmonitoring was carried out on a regular basis. Mice were injectedintrarenally on study day 0 with 1×10⁶ ACHN cells, randomized andweighed. One week after the tumor cell injection, mice were injectedi.v. with 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab three times a weekfor three weeks. All mice were injected i.v. with 200 μl of theappropriate solution. The mice in the vehicle group were injected withPBS and the treatment groups with 4G8 Fab-IL-2 wt-Fab or 4G8 Fab-IL-2qm-Fab immunoconjugate. To obtain the proper amount of immunoconjugateper 200 μl, the stock solutions were diluted with PBS when necessary.FIG. 25 shows that both 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fabimmunoconjugates mediated superior efficacy in terms of enhanced mediansurvival compared to vehicle group with an advantage for the 4G8Fab-IL-2 wt-Fab over the 4G8 Fab-IL-2 qm-Fab immunoconjugate in terms ofefficacy.

TABLE 14-A Concen- tration Compound Dose Formulation buffer (mg/mL) 4G8Fab-IL-2- 20 μg 25 mM potassium phosphate, 1.45 Fab wild type = 125 mMNaCl, FAP 4G8 wt 100 mM glycine, pH 6.7 4G8 Fab-IL-2- 20 μg 25 mMpotassium phosphate, 4.25 Fab quadruple 125 mM NaCl, mutant = FAP 100 mMglycine, pH 6.7 4G8 qm

LLC1 Lewis Lung Carcinoma Syngeneic Model

The FAP-targeted 4G8 Fab-IL-2 qm-Fab and 28H1 Fab-IL-2 qm-Fabimmunoconjugates were tested using the mouse Lewis lung carcinoma cellline LLC1, i.v. injected into Black 6 mice. The LLC1 Lewis lungcarcinoma cells were originally obtained from ATCC and after expansiondeposited in the Glycart internal cell bank. The tumor cell line wasroutinely cultured in DMEM containing 10% FCS (Gibco) at 37° C. in awater-saturated atmosphere at 5% CO₂. Passage 10 was used fortransplantation, at a viability of 97.9%. 2×10⁵ cells per animal wereinjected i.v. into the tail vein in 200 μl of Aim V cell culture medium(Gibco). Black 6 mice (Charles River, Germany), aged 8-9 weeks at thestart of the experiment, were maintained under specific-pathogen-freeconditions with daily cycles of 12 h light/12 h darkness according tocommitted guidelines (GV-Solas; Felasa; TierschG). The experimentalstudy protocol was reviewed and approved by local government (P2008016). After arrival, animals were maintained for one week to getaccustomed to the new environment and for observation. Continuous healthmonitoring was carried out on a regular basis. Mice were injected i.v.on study day 0 with 2×10⁵ of LLC1 cells, randomized and weighed. Oneweek after the tumor cell injection, mice were injected i.v. with 4G8Fab-IL-2qm-Fab or 28H1 Fab-IL-2 qm-Fab, three times a week for threeweeks. All mice were injected i.v. with 200 μl of the appropriatesolution. The mice in the vehicle group were injected with PBS and thetreatment group with the 4G8 Fab-IL-2 qm-Fab or 28H1 Fab-IL-2 qm-Fabconstructs. To obtain the proper amount of immunoconjugate per 200 μl,the stock solutions were diluted with PBS when necessary. FIG. 26 showsthat the 408 Fab-IL-2 qm-Fab or the affinity matured 28H1 Fab-IL-2qm-Fab constructs mediated superior efficacy in terms of enhanced mediansurvival compared to the vehicle group.

TABLE 14-B Concen- tration Compound Dose Formulation buffer (mg/mL) 28H1Fab-IL-2- 30 μg 25 mM potassium phosphate, 2.74 Fab quadruple 125 mMNaCl, mutant = FAP 100 mM glycine, pH 6.7 28H1 qm 4G8 Fab-IL-2- 30 μg 25mM potassium phosphate, 4.25 Fab quadruple 125 mM NaCl, mutant = FAP 100mM glycine, pH 6.7 4G8 qm

In another experiment, the FAP-targeted 28H1 Fab-IL-2 wt-Fab and 28H1Fab-IL-2 qm-Fab immunoconjugates were tested in the same mouse Lewislung carcinoma cell line LLC1, i.v. injected into Black 6 mice. Passage9 was used for transplantation, at a viability of 94.5%. 2×10⁵ cells peranimal were injected i.v. into the tail vein in 200 μl of Aim V cellculture medium (Gibco). Mice were injected i.v. on study day 0 with2×10⁵ of LLC1 cells, randomized and weighed. One week after the tumorcell injection, mice were injected i.v. with 28H1 Fab-IL-2 wt-Fab or28H1 Fab-IL-2 qm-Fab, three times a week for three weeks. All mice wereinjected i.v. with 200 μl of the appropriate solution. The mice in thevehicle group were injected with PBS and the treatment group with the28H1 Fab-IL-2 wt-Fab or 28H1 Fab-IL-2 qm-Fab constructs. To obtain theproper amount of immunoconjugate per 200 μl, the stock solutions werediluted with PBS when necessary. FIG. 27 shows that the 28H1 Fab-IL-2wt-Fab and 28H1 Fab-IL-2 qm-Fab immunoconjugates mediated superiorefficacy in terms of enhanced median survival compared to the vehiclegroup with a slight advantage for the 28H1 Fab-IL-2 wt-Fab over the 28H1Fab-IL-2 qm-Fab immunoconjugate in terms of efficacy.

TABLE 14-C Concen- tration Compound Dose Formulation buffer (mg/mL) 28H1Fab-IL-2- 45 μg 25 mM potassium phosphate, 2.74 Fab quadruple 125 mMNaCl, mutant = FAP 100 mM glycine, pH 6.7 28H1 qm 28H1 Fab-IL-2- 45 μg25 mM potassium phosphate, 1.66 Fab wild-type = 125 mM NaCl, FAP 28H1 wt100 mM glycine, pH 6.7

Example 9

The 4G8 based FAP-targeted Fab-IL-2 qm-Fab was subsequently compared tothe 4G8 based FAP-targeted Fab-IL-2 wt-Fab immunoconjugate in aseven-day intravenous toxicity and toxicokinetic study in Black 6 mice.Table 15 shows the study design of the toxicity and toxicokineticstudies.

TABLE 15 Study design. Dose Group Type [μg/g] Purpose 1 DPBS 0 Control 24G8 Fab-IL-2 wt-Fab 4.5 Toxicity titration 3 9.0 4 4G8 Fab-IL-2 qm-Fab4.5 5 9.0 6 4G8 Fab-IL-2 wt-Fab 4.5 Toxicokinetic 7 9.0 study 8 4G8Fab-IL-2 qm-Fab 4.5 9 9.0

The purpose of this study was to characterize and compare the toxicityand toxicokinetic profiles of FAP-targeted 4G8 Fab-IL2-Fab wild type(wt) interleukin-2 (IL-2) and FAP-targeted G48 Fab-IL-2-Fab quadruplemutant IL-2 (qm) after once daily intravenous administration tonon-tumor-bearing male mice for 7 days. For this study, 5 groups of 5male mice/group were administered intravenously 0 (vehicle control), 4.5or 9 μg/g/day wt IL-2, or 4.5 or 9 μg/g/day qm IL-2. An additional 4groups of 6 male mice/group were administered 4.5 or 9 μg/g/day wt IL-2,or 4.5 or 9 μg/day qm IL-2 in order to assess toxicokinetics. The studyduration was changed from 7 days to 5 days due to clinical signsobserved in animals given 4.5 and 9 μg/g/day wt IL-2. Assessment oftoxicity was based upon mortality, in-life observations, body weight,and clinical and anatomic pathology. Blood was collected at various timepoints from animals in the toxicokinetic groups for toxicokineticanalysis. The toxicokinetic data showed that the mice treated with wtIL-2 or qm IL-2 had measurable plasma levels up to the last bleedingtime, indicating that the mice were exposed to the respective compoundsthroughout the duration of treatment. Day 1 AUC0-inf values suggestcomparable exposure of wt IL-2 and qm IL-2 at both dose levels. Sparsesamples were taken on Day 5 and showed equivalent plasma concentrationsto Day 1, suggesting no accumulation occurred after 5 days of dosingeither compound. In more details the following findings were observed.

Toxicokinetics

Table 16 summarizes the mean plasma toxicokinetic parameters for theFAP-targeted 4G8 Fab-IL-2 qm-Fab and the FAP-targeted 4G8 Fab-IL-2wt-Fab as determined by WinNonLin Version 5.2.1 and a commercialkappa-specific ELISA (Human Kappa ELISA Quantitation Set, BethylLaboratories).

TABLE 16 Group 6 Group 7 Group 8 Group 9 4G8-FAP-Wild 4G8-FAP-Wild4G8-FAP- 4G8-FAP- Parameter Units Type IL-2 Type IL-2 Mutant IL-2 MutantIL-2 Cmax ng/ml 47198 97986 60639 146415 Cmax/Dose (ng/ml)/(ug/g) 0.0110.011 0.0135 0.016 AUC ng*h/ml 331747 747449 355030 926683 AUC/Dose(ng*h/ml)/(ug/g) 0.074 0.083 0.079 0.103 T½z h 3.6 3.11 4.3 3.12Original Dose ug/g 4.5 9 4.5 9 Route IV IV IV IV *TK Parameters werecalculated in WinNonlin Version 5.2.1 using noncompartmental analysis

The individual serum concentrations are given in the following:

Serum Mean Bleed Time conc. conc Group (dose) Day (h) Animal (ng/ml)(ng/ml) Group 6 1 1 26 64241 47198 (4.5 μg/g) 27 30155 4G8 Fab-II2- 15.5 28 14693 15784 Fab WT 29 16875 1 24 30 318 419 31 520 5 5.5 29 1306113335 30 13620 31 13325 Group 7 1 1 32 101208 97986 (9 μg/g) 33 947644G8 Fab-II2- 1 5.5 34 35766 34062 Fab WT 35 32359 1 24 36 573 580 37 5885 5.5 32 31779 37473 33 51143 35 53409 36 13562 Group 8 1 1 38 7332660639 (4.5 μg/g) 39 47953 4G8 Fab-II2- 1 5.5 40 12168 13269 Fab Mutant41 14371 1 24 42 494 490 43 487 5 5.5 40 6561 10957 41 15352 5 24 38 608721 39 543 42 1298 43 437 Group 9 1 1 44 162970 146416 (9 μg/g) 45129862 4G8 Fab-II2- 1 5.5 46 20475 24800 Fab Mutant 47 29125 1 24 48 478493 49 509 5 5.5 48 20504 48031 47 75557 5 24 44 634 703 45 796 48 66149 719

These data show that both, the 4G8 Fab-IL-2 qm-Fab and the 4G8 Fab-IL-2wt-Fab show comparable pharmacokinetic properties with slightly higherexposure for the 4G8 Fab-IL-2 qm-Fab.

Mortality

In the 9 μg/g FAP-targeted 4G8 Fab-IL-2 wt-Fab group, treatment-relatedmortality occurred in one animal prior to necropsy on Day 5.Hypoactivity, cold skin, and hunched posture were noted prior to death.This animal likely died due to a combination of cellular infiltration inthe lung that was accompanied with edema and hemorrhage and marked bonemarrow necrosis. Mortality is summarized in Table 17.

TABLE 17 Mortality day 5. Severe Dose Found toxicity Group Type [μg/g]dead Sacrifice** Total 1 DPBS 0 0/5 0/5 0/5 2 4G8 Fab-IL-2 wt-Fab 4.50/5 5/5 5/5 3 9  1/5* 4/5 4/5 4 4G8 Fab-IL-2 qm-Fab 4.5 0/5 0/5 0/5 5 90/5 0/5 0/5 6 4G8 Fab-IL-2 wt-Fab 4.5 1/6 5/6 6/6 7 9 2/6 4/6 6/6 8 4G8Fab-IL-2 qm-Fab 4.5 0/6 0/6 0/6 9 9 0/6 0/6 0/6 *in route to necropsy**study was planned for seven days but all mice treated with thewild-type IL-2 immunoconjugate were markedly affected by Day 5 and weresacrificed as they were not expected to survive.

Clinical Observations

Observations of hypoactivity, cold skin, and hunched posture were notedin animals given 4.5 and 9 μg/g/day wt IL-2. Clinical observations aresummarized in Table 18.

TABLE 18 Clinical observations day 5. Dose Hunched Hypo- Cool to GroupType [μg/g] posture active touch 1 DPBS 0 0/5 0/5 0/5 2 4G8 Fab-IL-2wt-Fab 4.5 4/5 4/5 5/5 3 9 5/5 5/5 5/5 4 4G8 Fab-IL-2 qm-Fab 4.5 0/5 0/50/5 5 9 0/5 0/5 0/5 6 4G8 Fab-IL-2 wt-Fab 4.5 6/6 2/6 2/6 7 9 6/6 5/66/6 8 4G8 Fab-IL-2 qm-Fab 4.5 0/6 0/6 0/6 9 9 0/6 0/6 0/6

Body Weight

A moderate decrease in body weight was observed after 5 days oftreatment in animals given 4.5 and 9 (9% and 11%, respectively) μg/g/daywt IL-2. A slight decrease in body weight was observed after 5 days oftreatment in animals given 4.5 or 9 (2% and 1%, respectively) μg/g/dayqm IL-2. A moderate (9%) decrease in body weight was also observed invehicle controls after 5 days of treatment. However, the percentdecrease would have been 5% if a potential outlier (Animal #3) wasexcluded. The body weight loss in the vehicle group may have beenattributed to stress.

Hematology

A reduced platelet count was observed in animals given 4.5 (˜4.5 fold)and 9 μg/g/day (11 fold) 4G8 Fab-IL-2 wt-Fab, which correlated withreduced megakaryocytes in the bone marrow as well as systemicconsumptive effects (fibrin) in spleen and lung of these animals (seeHistopathology section below) These findings indicated that reducedplatelets were likely due to combined effects of consumption anddecrease in production/bone marrow crowding due to increase inlymphocyte/myeloid cell production as a direct or indirect effect ofIG-2.

Hematologic findings of uncertain relationship to compoundadministration consisted of absolute lymphocyte count decreases with 4G8Fab-IL-2 wt-Fab at 4.5 (˜5-fold) and 9 μg/g (˜3-fold) compared to themean value of the vehicle control group. These findings lacked cleardose-dependency, but could be considered secondary to effects associatedwith stress noted in in-life observations or exaggerated pharmacology ofthe compound (lymphocytes migrating into tissues). There were notreatment-related hematological changes attributed to the administrationof 4G8-Fab-IL-2 qm-Fab. A few isolated hematologic findings werestatistically different from their respective controls. However, thesefindings were of insufficient magnitude to suggest pathologicalrelevance.

Gross Pathology and Histopathology

Treatment-related gross findings included enlarged spleen found in 5/5and 4/5 mice of 4.5 and 9 μg/g 4G8 Fab-IL-2 wt-Fab groups, respectively,and in 1/5 in both 4.5 and 9 μg/g 4G8 Fab-IL-2 qm-Fab treatment groups.

Treatment-related histopathology findings were present in groups given4.5 and 9 μg/g 408 Fab-IL-2 wt-Fab and 4.5 and 9 μg/g 4G8 Fab-IL-2qm-Fab in lung, bone marrow, liver, spleen, and thymus, with differencesin incidence, severity grading or nature of the changes, as reportedbelow.

Treatment-related histopathology findings in the lung consisted ofmononuclear infiltration found mild to marked in 5/5 mice of the 4.5 and9 μg/g 408 Fab-IL-2 wt-Fab groups and marginally in 5/5 mice of the 4.5and 9 μg/g 4G8 Fab-IL-2 qm-Fab groups. Mononuclear infiltrationconsisted of lymphocytes (some of which were noted as having cytoplasmicgranules) as well as reactive macrophages. These cells were most oftennoted to have vasocentric patterns, often with margination noted withinthe vessels in the lung. These cells were also noted surrounding thevessels, but in more severe cases, the pattern was more diffuse.Hemorrhage was seen marginal to mild in 5/5 mice of the 4.5 and 9 μg/g4G8 Fab-IL-2 wt-Fab groups and marginally in 2/5 mice in the 9 μg/g 4G8Fab-IL-2.qm-Fab group. Though the hemorrhage was most often notedperivascularly, in more severe cases, it was noted in alveolar spaces.Edema was noted mild to moderate in 5/5 mice in the 4.5 and 9 μg/g 4G8Fab-IL-2 wt-Fab groups and marginally in 5/5 mice in the 9 g/g 4G8Fab-IL-2 qm-Fab group. Though the edema was frequently seenperivascularly, in more severe cases, it was noted in alveolar spaces aswell. Marginal cellular degeneration and karyorrhexis was noted in 2/5and 5/5 mice in the 4.5 and 9 μg/g 4G8 Fab-IL-2 wt-Fab groups,respectively and consisted of degeneration of infiltrative or reactiveleukocytes. Selected animals with MSB stains were positive for fibrinfound within the lungs of animals in both 4.5 and 9 μg/g 4G8 Fab-IL-2wt-Fab groups which correlates in part with the reduced platelets notedin these animals.

Treatment-related changes in the bone marrow included marginal to mildincreased overall marrow cellularity in 5/5 mice and 2/5 mice of both4.5 and 5/5 mice and 2/5 mice of both 9 μg/g 4G8 Fab-IL-2 wt-Fab and 4G8Fab-IL-2 qm-Fab groups, respectively. This was characterized byincreased marginal to moderate lymphocyte-myelocyte hyperplasia in thesegroups that was supported, in part, by increased numbers of CD3 positiveT cells within the marrow and sinuses (specifically T-lymphocytes,confirmed by immunohistochemistry with the pan-T-cell marker CD3 done onselected animals). CD3 positive T cell increase was moderate in both 4G8Fab-IL-2 wt-Fab groups and marginal to mild in both 4G8 Fab-IL-2 qm-Fabgroups Marginal to mild decreases in megakaryocytes were observed in 2/5mice in the 4.5 and 5/5 mice in the 9 μg/g 4G8 Fab-IL-2 wt-Fab groupsand marginal to moderate decreases in erythroid precursors were noted in3/5 mice in the 4.5 and 5/5 mice in the 9 μg/g 4G8 Fab-IL-2 wt-Fabgroups. Bone marrow necrosis was noted in 1/5 mice in 4.5 (minimal) and5/5 mice in 9 (mild to marked) μg/g 4G8 Fab-IL-2 wt-Fab groups. Thereduced number of megakaryocytes in the bone marrow correlated withdecreased platelets which could be due to direct crowding of the bonemarrow by increased lymphocytes/myeloid precursors and/or the bonemarrow necrosis, and/or consumption of platelets due to inflammation invarious tissues (see spleen and lung). The decreased erythroidprecursors noted in the bone marrow, did not correlate with theperipheral blood hematology findings likely due to temporal effects(seen in bone marrow before peripheral blood) and the longer half-lifeof peripheral erythrocytes (compared to platelets). The mechanism ofbone marrow necrosis in the bone marrow may be secondary due to overtovercrowding of the marrow cavity (due to production and growth oflymphocytes/myeloid cells), systemic or local release of cytokines fromthe proliferating cell types, possibly related to local affects ofhypoxia or other pharmacologic effects of the compound.

Treatment-related findings in the liver consisted of mild to moderateprimarily vasocentric mononuclear cell infiltrate and marginal to mildsingle cell necrosis in 5/5 mice of the 4.5 and 9 μg/g 4G8 Fab-IL-2wt-Fab groups. Marginal single cell necrosis was seen in 2/5 and 4/5mice in the 4.5 and 9 μg/g 4G8 Fab-IL-2 qm-Fab groups, respectively. Themononuclear infiltrate consisted primarily of lymphocytes (specificallyT-lymphocytes, confirmed by immunohistochemistry with the pan-T cellmarker CD3 done on selected animals) that were most often notedvasocentrically as well as marginating within the central and portalvessels. Selected animals for immunohistochemistry staining for F4/80showed increased numbers and size (activated) of macrophages/Kupffercells throughout the hepatic sinusoids in 9 μg/g 4G8 Fab-IL-2 wt-Fab and4G8 Fab-IL-2 qm-Fab groups.

Treatment-related findings in the spleen consisted of moderate to markedlymphoid hyperplasia/infiltration and mild to moderate macrophagehyperplasia/infiltration in 5/5 mice in 4.5 and 9 μg/g 408 Fab-IL-2wt-Fab groups and mild to moderate lymphoid hyperplasia/infiltrationwith marginal to mild macrophage hyperplasia/infiltration in 5/5 mice in4.5 and 9 μg/g 4G8 Fab-IL-2 qm-Fab groups. Immunohistochemistry for 9μg/g 4G8 Fab-IL-2 wt-Fab and 4G8 Fab-IL-2 qm-Fab showed differentpatterns using the pan-T cell marker CD3, as well as the macrophagemarker F4/80. For 9 μg/g 4G8 Fab-IL-2 wt-Fab, the pattern of T-cell andmacrophage immunoreactivity remained primarily within the red pulpareas, as the architecture of the primary follicles had been altered bylymphocytolysis and necrosis (described below). For 9 μg/g 4G8 Fab-IL-2qm-Fab, special stains showed a pattern similar to that of the vehiclecontrol, but with periarteriolar lymphoid sheath (PALS) white pulpexpansion, by a T-cell population and a larger, expanded red pulp area.T-cell and macrophage positivity was also evident within the red pulp,with a similar pattern to the vehicle control group, but expanded. Thesefindings correlate with the gross findings of enlarged spleen. Necrosiswas noted marginally in 3/5 mice and marginally to mildly in 5/5 mice in4.5 and 9 μg/g 4G8 Fab-IL-2 wt-Fab groups, respectively. Necrosis wasusually located around the area of the primary follicles and selectedanimals using MSB stain were positive for fibrin in both 4.5 and 9 μg/g4G8 Fab-IG-2 wt-Fab groups which correlates in part with the reducedplatelets noted in these animals. Lymphocytolysis was seen in the 4.5μg/g (minimal to mild) and 9 μg/g (moderate to marked) 4G8 Fab-IL-2wt-Fab groups.

Treatment-related findings in the thymus included minimal to mildincreases in lymphocytes in both 4.5 and 9 μg/g 4G8 Fab-IL-2 wt-Fab andin 4.5 ug/g 4G8 Fab-IL-2 qm-Fab groups. The cortex and medulla were notindividually evident, in 4G8 Fab-IL-2 wt-Fab groups, butimmunohistochemistry for the pan T cell marker (CD3) on selected animalsin 9 μg/g 4G8 Fab-IL-2 wt-Fab and 9 μg/g 4G8 Fab-IL-2 qm-Fab groupsshowed strong positivity for the majority of the cells within thethymus. Increased lymphocytes in the thymus was considered to be adirect pharmacologic effect of both compounds where IL-2 inducedproliferation of lymphocytes migrating to the thymus (T cells) from thebone marrow for further differentiation and clonal expansion. Thisoccurred in all groups except 9 μg/g 4G8 Fab-IL-2 qm-Fab, which islikely a temporal effect. Lymphocytolysis was mild in 4.5 μg/g 4G8Fab-IL-2 wt-Fab group, and was moderate to marked in the 9 μg/g 4G8Fab-IL-2 wt-Fab group. Moderate lymphoid depletion was noted in both 4.5and 9 μg/g 4G8 Fab-IL-2 wt-Fab groups. While these findings appear morerobust in the 4.5 and 9 μg/g 4G8 Fab-IL-2 wt-Fab groups, these animalswere described as moribund on Day 5, and the mild to markedlymphocytolysis as well as moderate lymphoid depletion may be related tothis in-life observation (stress-related effects due to poor physicalcondition).

Histopathology findings of uncertain relationship to compoundadministration in the liver consisted of a marginal mixed cell(lymphocytes and macrophages) infiltrate/activation noted as smallfoci/microgranulomas scattered randomly throughout the liver in 5/5 micein both 4.5 and 9 μg/g 4G8 Fab-IL-2 qm-Fab groups. This marginal changewas also seen in the vehicle control group but with fewer incidence andseverity. Stomach glandular dilation and atrophy was seen marginally tomildly in 5/5 mice and ileal villous atrophy was seen marginally in 3/5mice in the 9 μg/g 4G8 Fab-IL-2 wt-Fab group. This finding is mostlikely attributed to poor physical condition seen in these mice such asreduced body weight, especially in the 9 μg/g 4G8 Fab-IL-2 wt-Fab groupnoted in the in-life observations.

Injection site findings included mixed cell infiltrate, perivascularedema, and myodegeneration that was noted equally in vehicle control, 9μg/g 4G8 Fab-IL-2 wt-Fab and 9 μg/g 4G8 Fab-IL-2 qm-Fab groups. Oneanimal had epidermal necrosis. These findings were not attributed to thetreatment(s) itself, but to the daily i.v. injection and handling of thetail. Another animal had macrophage infiltration of the skeletal muscle(noted on the lung tissue histology section) associated withmyodegeneration and myodegeneration likely due to a chronic lesion andwas not attributed to the treatment. Marginal lymphoid depletion wasnoted in 3/5 and 4/5 mice in the 4.5 and 9 μg/g 4G8 Fab-IL-2 qm-Fabgroups, respectively and was most likely attributed to normalphysiologic changes seen in the thymus as mice get older (also seen insimilar incidence, 4/5 mice, and severity in vehicle control animals).

In conclusion, the daily intravenous administration of 4G8 Fab-IL-2wt-Fab or 4G8 Fab-IL-2 qm-Fab at doses of 4.5 or 9 μg/g/day for up to 5days in male mice resulted in similar treatment-related histologicfindings with both compounds. However, the findings were generally moreprevalent and more severe with FAP-targeted 4G8 Fab-IL-2 wt-Fab in thelung (FIGS. 28 and 29) (mononuclear infiltration consisting oflymphocytes and reactive macrophages, hemorrhage, and edema), bonemarrow (lympho-myelo hyperplasia and increased cellularity), liver (FIG.30) (single cell necrosis, Kupffer cell/macrophage increase in numberand activation), spleen (grossly enlarged, macrophage and lymphocyteinfiltration/hyperplasia) and thymus (increased lymphocytes). Inaddition, mortality, lymphocytolysis, necrosis or cellular degenerationin the lung spleen, bone marrow, and thymus, as well as reducedmegakaryocytes and erythrocytes in bone marrow and reduced platelets inperipheral blood were seen only in animals given wt IL-2.

Based on the clinical and anatomic pathologic findings, as well asclinical observations, and the comparable systemic exposure of bothcompounds, the qm IL-2 under conditions of this study exhibited markedlyless systemic toxicity following 5 doses than wt IL-2.

Example 10

Induction of NK Cell IFN-γ Secretion by Wild Type and Quadruple MutantIL-2

NK-92 cells were starved for 2 h before seeding 100000 cells/well into a96 well-F-bottom plate. IL-2 constructs were titrated onto the seededNK-92 cells. After 24 h or 48 h, plates were centrifuged beforecollecting the supernatants to determine the amount of human IFN-γ usinga commercial IFN-γ ELISA (BD #550612).

Two different in-house preparations of wild type IL-2 (probablydiffering slightly in their 0-glycosylation profiles, see Example 2), acommercially available wild-type IL-2 (Proleukin) and in-house preparedquadruple mutant IL-2 (first batch) were tested.

FIG. 31 shows that the quadruple mutant IL-2 is equally potent ascommercially obtained (Proleukin) or in-house produced wild-type IL-2 ininducing IFN-γ secretion by NK cells for 24 (A) or 48 hours (B).

Example 11

Induction of NK Cell Proliferation by Wild Type Ad Quadruple Mutant IL-2

NK-92 cells were starved for 2 h before seeding 10000 cells/well into96-well-black-F-clear bottom plates. IL-2 constructs were titrated ontothe seeded NK-92 cells. After 48 h the ATP content was measured todetermine the number of viable cells using the “CellTiter-GloLuminescent Cell Viability Assay” Kit from Promega according to themanufacturer's instructions.

The same IL-2 preparations as in Example 10 were tested.

FIG. 32 shows that all tested molecules were able to induceproliferation of NK cells. At low concentrations (<0.01 nM) thequadruple mutant IL-2 was slightly less active than the in-houseproduced wild-type IL-2, and all in-house preparations were less activethan the commercially obtained wild-type IL-2 (Proleukin).

In a second experiment, the following IL-2 preparations were tested:wild-type IL2 (pool 2), quadruple mutant IL-2 (first and second batch).

FIG. 33 shows that all tested molecules were about similarly active ininducing proliferation of NK cells, with the two mutant IL-2preparations being only minimally less active than the wild-type IL-2preparations at the lowest concentrations.

Example 12

Induction of human PBMC proliferation by Immunoconjugates comprisingwild type or quadruple mutant IL-2

Peripheral blood mononuclear cells (PBMC) were prepared usingHistopaque-1077 (Sigma Diagnostics Inc., St. Louis, Mo., USA). In brief,venous blood from healthy volunteers was drawn into heparinizedsyringes. The blood was diluted 2:1 with calcium- and magnesium-freePBS, and layered on Histopaque-1077. The gradient was centrifuged at450×g for 30 min at room temperature (RT) without breaks. The interphasecontaining the PBMCs was collected and washed three times with PBS(350×g followed by 300×g for 10 min at RT).

Subsequently, PBMCs were labeled with 40 nM CFSE (carboxyfluoresceinsuccinimidyl ester) for 15 min at 37° C. Cells were washed with 20 mlmedium before recovering the labeled PBMCs for 30 min at 37° C. Thecells were washed, counted, and 100000 cells were seeded into96-well-U-bottom plates. Pre-diluted Proleukin (commercially availablewild-type IL-2) or IL2-immunoconjugates were titrated onto the seededcells which were incubated for the indicated time points. After 4-6days, cells were washed, stained for appropriate cell surface markers,and analyzed by FACS using a BD FACSCantoII. NK cells were defined asCD3/CD56⁺, CD4⁺ T cells as CD3⁺/CD8⁻ and CD8 T cells as CD3⁺/CD8⁺.

FIG. 34 shows proliferation of NK cells after incubation with differentFAP-targeted 28H1 IL-2 immunoconjugates for 4 (A), 5 (B) or 6 (C) days.All tested constructs induced NK cell proliferation in aconcentration-dependent manner. Proleukin was more efficacious than theimmunoconjugates at lower concentrations, this difference no longerexisted at higher concentrations, however. At earlier time points (day4); the IgG-1L2 constructs appeared slightly more potent than theFab-IL2-Fab constructs. At later time points (day 6), all constructs hadcomparable efficacy, with the Fab-IL2 qm-Fab construct being leastpotent at the low concentrations.

FIG. 35 shows proliferation of CD4 T-cells after incubation withdifferent FAP-targeted 28H1 IL-2 immunoconjugates for 4 (A), 5 (B) or 6(C) days. All tested constructs induced CD4 T cell proliferation in aconcentration-dependent manner. Proleukin had a higher activity than theimmunoconjugates, and the immunoconjugates comprising wild-type IL-2were slightly more potent than the ones comprising quadruple mutantIL-2. As for the NK cells, the Fab-IL2 qm-Fab construct had the lowestactivity. Most likely the proliferating CD4 T cells are partlyregulatory T cells, at least for the wild-type IL-2 constructs.

FIG. 36 shows proliferation of CD8 T-cells after incubation withdifferent FAP-targeted 28H1 IL-2 immunoconjugates for 4 (A), 5 (B) or 6(C) days. All tested constructs induced CD8 T cell proliferation in aconcentration-dependent manner. Proleukin had a higher activity than theimmunoconjugates, and the immunoconjugates comprising wild-type IL-2were slightly more potent than the ones comprising quadruple mutantIL-2. As for the NK and CD4 T cells, the Fab-IL2 qm-Fab construct hadthe lowest activity.

FIG. 37 depicts the results of another experiment, wherein FAP-targeted28H1 IgG-IL-2, comprising either wild-type or quadruple mutant IL-2, andProleukin were compared. Incubation time was 6 days. As shown in thefigure, all three IL-2 constructs induce NK (A) and CD8 T-cell (C)proliferation in a dose-dependent manner with similar potency. For CD4T-cells (B), the IgG-IL2 qm immunoconjugate has a lower activity,particularly at medium concentrations, which might be due to its lack ofactivity on CD25-positive (including regulatory) T cells which are asubset of CD4 T cells.

Example 13

Effector Cell Activation by Wild-Type and Quadruple Mutant IL-2 (pSTAT5Assay)

PBMCs were prepared as described above. 500000 PBMCs/well were seededinto 96-well-U-bottom plates and rested 45 min at 37° C. in RPMI mediumcontaining 10% FCS and 1% Glutamax (Gibco). Afterwards, PBMCs wereincubated with Proleukin, in-house produced wild-type IL-2 or quadruplemutant IL-2 at the indicated concentrations for 20 min at 37° C. toinduce phosphorylation of STAT5. Subsequently, cells were immediatelyfixed (BD Cytofix Buffer) for 10 min at 37° C. and washed once, followedby a permeabilization step (BD Phosflow Perm Buffer III) for 30 min at4° C. Afterwards, cells were washed with PBS/0.1% BSA and stained withmixtures of FACS antibodies for detection of NK cells (CD3⁻/CD56⁺), CD8⁺T cells (CD3⁺/CD8⁺), CD4⁺ T cells (CD3⁺/CD4⁺/CD25⁻/CD127⁺) or T_(reg)cells (CD4⁺/CD25⁺/CD127⁻/FoxP3⁺), as well as pSTAT5 for 30 min at RT inthe dark. Cells were washed twice with PBS/0.1% BSA and resuspended in2% PFA before flow cytometric analysis (BD FACSCantoII). FIG. 38 showsSTAT phosphorylation in NK cells (A), CD8 T-cells (B), CD4 T-cells (C)and regulatory T-cells (D) after 30 min incubation with Proleukin,in-house produced wild-type IL-2 (pool 21 and quadruple mutant IL-2(batch 1). All three IL-2 preparations were equally potent in inducingSTAT phosphorylation in NK as well as CD8 T-cells. In CD4 T-cells andeven more so in regulatory T-cells, the quadruple mutant IL-2 had alower activity than the wild-type IL-2 preparations.

Example 14

Effector Cell Activation by Wild-Type and Quadruple Mutant IgG-IL-2(pSTAT5 Assay)

Experimental conditions were as described above (see Example 13).

FIG. 39 shows STAT phosphorylation in NK cells (A), CD8 T-cells (B), CD4T-cells (C) and regulatory T-cells (D) after 30 min incubation withProleukin, IgG-IL-2 comprising wild-type IL-2 or IgG-IL-2 comprisingquadruple mutant IL-2. On all cell types Proleukin was more potent ininducing STAT phosphorylation than the IgG-IL-2 immunoconjugates. TheIgG-IL-2 wild-type and quadruple mutant constructs were equally potentin NK as well as CD8 T-cells. In CD4 T-cells and even more so inregulatory T-cells, the IgG-IL-2 quadruple mutant had a lower activitythan the IgG-IL-2 wild-type immunoconjugate.

Example 15

Maximum Tolerated Dose (MTD) of FAP-Targeted Fab-IL2 wt-Fab and Fab-IL2qm-Fab Immunoconjugates

Escalating doses of FAP-targeted Fab-IL2-Fab immunoconjugates,comprising either wild type (wt) or quadruple mutant (qm) IL-2, weretested in tumor free immunocompetent Black 6 mice. Female Black 6 mice(Charles River, Germany), aged 8-9 weeks at the start of the experiment,were maintained under specific-pathogen-free conditions with dailycycles of 12 h light/12 h darkness according to committed guidelines(GV-Solas; Felasa; TierschG). The experimental study protocol wasreviewed and approved by local government (P 2008016). After arrival,animals were maintained for one week to get accustomed to the newenvironment and for observation. Continuous health monitoring wascarried out on a regular basis.

Mice were injected i.v. once a day for 7 days with 4G8 Fab-IL2 wt-Fab atdoses of 60, 80 and 100 μg/mouse or 4G8 Fab-IL2 qm-Fab at doses of 100,200, 400, 600 and 1000 μg/mouse. All mice were injected i.v. with 200 μlof the appropriate solution. To obtain the proper amount ofimmunoconjugate per 200 μl, the stock solutions were diluted with PBS asnecessary.

FIG. 40 shows that the MTD (maximum tolerated dose) for Fab-1L2 qm-Fabis 10-fold higher than for Fab-IL2 wt-Fab, namely 600 μg/mouse daily for7 days for the Fab-IL2 qm-Fab vs. 60 μg/mouse daily for 7 days for theFab-IL2 wt-Fab.

TABLE 19 Concen- tration Compound Dose Formulation buffer (mg/mL) 4G8Fab- 60, 80, 25 mM potassium phosphate, 3.32 IL2 wt-Fab 100 μg 125 mMNaCl, (=stock 100 mM glycine, pH 6.7 solution) 4G8 Fab- 100, 200, 25 mMpotassium phosphate, 4.25 IL2 qm-Fab 400, 600, 125 mM NaCl, (=stock 1000μg 100 mM glycine, pH 6.7 solution)

Example 16

Pharmacokinetics of a Single Dose of FAP-Targeted and Untargeted IgG-IL2wt and Qm

A single dose pharmacokinetics (PK) study was performed in tumor-freeimmunocompetent 129 mice for FAP-targeted-IgG-IL2 immunoconjugatescomprising either wild type or quadruple mutant IL-2, and untargetedIgG-IL2 immunoconjugates comprising either wild type or quadruple mutantIL-2.

Female 129 mice (Harlan, United Kingdom), aged 8-9 weeks at the start ofthe experiment, were maintained under specific-pathogen-free conditionswith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (P 2008016). Afterarrival, animals were maintained for one week to get accustomed to thenew environment and for observation. Continuous health monitoring wascarried out on a regular basis.

Mice were injected i.v. once with FAP-targeted 28H1 IgG-IL2 wt (2.5mg/kg) or 28H1 IgG-IL2 qm (5 mg/kg), or untargeted DP47GS IgG-IL2 wt (5mg/kg) or DP47GS IgG-IL2 qm (5 mg/kg). All mice were injected i.v. with200 μl of the appropriate solution. To obtain the proper amount ofimmunoconjugate per 200 μl, the stock solutions were diluted with PBS asnecessary. Mice were bled at 1, 8, 24, 48, 72, 96 h; and every 2 daysthereafter for 3 weeks. Sera were extracted and stored at −20° C. untilELISA analysis. Immunoconjugate concentrations in serum were determinedusing an ELISA for quantification of the IL2-immunoconjugate antibody(Roche-Penzberg). Absorption was measured using a measuring wavelengthof 405 nm and a reference wavelength of 492 nm (VersaMax tunablemicroplate reader, Molecular Devices).

FIG. 41 shows the pharmacokinetics of these IL-2 immunoconjugates. Boththe FAP-targeted (A) and untargeted (B) IgG-IL2 qm constructs have alonger serum half-life (approx. 30 h) than the corresponding IgG-IL2 wtconstructs (approx. 15 h).

TABLE 20 Concen- tration Compound Dose Formulation buffer (mg/mL)28H1-IgG- 2.5 mg/kg   20 mM Histidine, 3.84 IL2 wt 140 mM NaCl, pH 6.0(=stock solution) 28H1-IgG- 5 mg/kg 20 mM Histidine, 2.42 IL2 qm 140 mMNaCl, pH 6.0 (=stock solution) DP47GS- 5 mg/kg 20 mM Histidine, 3.74IgG-IL2wt 140 mM NaCl, pH 6.0 (=stock solution) DP47GS- 5 mg/kg 20 mMHistidine, 5.87 IgG- 140 mM NaCl, pH 6.0 (=stock IL2QM solution)

Example 17

Pharmacokinetics of a Single Dose of Untargeted Fab-IL2 wt-Fab andFab-IL2 qm-Fab

A single dose pharmacokinetics (PK) study was performed in tumor-freeimmunocompetent 129 mice for untargeted Fab-IL2-Fab immunoconjugatescomprising either wild type or quadruple mutant IL-2.

Female 129 mice (Harlan, United Kingdom), aged 8-9 weeks at the start ofthe experiment, were maintained under specific-pathogen-free conditionswith daily cycles of 12 h light/12 h darkness according to committedguidelines (GV-Solas; Felasa; TierschG). The experimental study protocolwas reviewed and approved by local government (P 2008016). Afterarrival, animals were maintained for one week to get accustomed to thenew environment and for observation. Continuous health monitoring wascarried out on a regular basis.

Mice were injected i.v. once with DP47GS Fab-IL2 wt-Fab at a dose of 65nmol/kg or DP47GS Fab-IL2 qm-Fab at a dose of 65 nM/kg. All mice wereinjected i.v. with 200 μl of the appropriate solution. To obtain theproper amount of immunoconjugate per 200 μl, the stock solutions werediluted with PBS as necessary.

Mice were bled at 0.5, 1, 3, 8, 24, 48, 72, 96 hours and thereafterevery 2 days for 3 weeks. Sera were extracted and stored at −20° C.until ELISA analysis. Immunoconjugate concentrations in serum weredetermined using an ELISA for quantification of IL2-immunoconjugateantibody (Roche-Penzberg). Absorption was measured using a measuringwavelength of 405 nm and a reference wavelength of 492 nm (VersaMaxtunable microplate reader, Molecular Devices).

FIG. 42 shows the pharmacokinetics of these IL-2 immunoconjugates.Fab-IL2-Fab wt and qm constructs have an approx. serum half-life of 3-4h. The difference in serum half-life between constructs comprisingwild-type or quadruple mutant IL-2 is less pronounced for theFab-1L2-Fab constructs than for IgG-like immunoconjugates, which per sehave longer half-lives.

TABLE 21 Concen- tration Compound Dose Formulation buffer (mg/mL) DP47GS65 nM/kg 100 mM glycine, 3.84 Fab-IL2 wt- 125 mM NaCl, (=stock Fab 25 mMKH₂PO₄, pH 6.7 solution) DP47GS 65 nM/kg 100 mM glycine, 2.42 Fab-IL2125 mMNaCl, (=stock qm-Fab 25 mM KH₂PO₄, pH 6.7 solution)

Example 18

Activation Induced Cell Death of IL-2 Activated PBMCs

Freshly isolated PBMCs from healthy donors were pre-activated overnightwith PHA-M at 1 μg/ml in RPMI1640 with 10% FCS and 1% Glutamine. Afterpre-activation PBMCs were harvested, labeled with 40 nM CFSE in PBS, andseeded in 96-well plates at 100 000 cells/well. Pre-activated PBMCs werestimulated with different concentrations of IL-2 immunoconjugates (4B9IgG-IL-2 wt, 4B9 IgG-IL-2 qm, 4B9 Fab-IL-2 wt-Fab, and 4B9 Fab-IL-2qm-Fab). After six days of IL-2 treatment PBMCs were treated with 0.5μg/ml activating anti-Fas antibody overnight. Proliferation of CD4(CD3⁺CD8⁻) and CD8 (CD3⁺CD8⁺) T cells was analyzed after six days byCFSE dilution. The percentage of living T cells after anti-Fas treatmentwas determined by gating on CD3⁺ Annexin V negative living cells.

As shown in FIG. 44, all constructs induced proliferation ofpre-activated T cells. At low concentrations the constructs comprisingwild-type IL-2 wt were more active than the IL-2 qm-comprisingconstructs. IgG-IL-2 wt, Fab-IL-2 wt-Fab and Proleukin had similaractivity. Fab-IL-2 qm-Fab was slightly less active than IgG-IL-2 qm. Theconstructs comprising wild-type IL-2 were more active on CD4 T cellsthan on CD8 T cells, most probably because of the activation ofregulatory T cells. The constructs comprising quadruple mutant IL-2 weresimilarly active on CD8 and CD4 T cells.

As shown in FIG. 45, T cells stimulated with high concentrations ofwild-type IL-2 are more sensitive to anti-Fas induced apoptosis than Tcells treated with quadruple mutant IL-2.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A mutant interleukin-2 (IL-2) polypeptide comprising at a first aminoacid mutation that abolishes or reduces affinity of the mutant IL-2polypeptide to the high-affinity IL-2 receptor and preserves affinity ofthe mutant IL-2 polypeptide to the intermediate-affinity IL-2 receptor,each compared to a wild-type IL-2 polypeptide, characterized in thatsaid first amino acid mutation is at a position corresponding to residue72 of human IL-2.
 2. The mutant interleukin-2 polypeptide of claim 1,wherein said first amino acid mutation is an amino acid substitution,selected from the group of L72G, L72A, L72S, L72T, L72Q, L72E, L72N,L72D, L72R, and L72K.
 3. The mutant interleukin-2 polypeptide of claim 1or 2, comprising a second amino acid mutation that abolishes or reducesaffinity of the mutant IL-2 polypeptide to the high-affinity IL-2receptor and preserves affinity of the mutant IL-2 polypeptide to theintermediate-affinity IL-2 receptor, each compared to a wild-type IL-2polypeptide.
 4. The mutant interleukin-2 polypeptide of claim 3, whereinsaid second amino acid mutation is at a position selected from thepositions corresponding to residue 35, 38, 42, 43, and 45 of human IL-2.5. The mutant interleukin-2 polypeptide of claim 3 or 4, wherein saidsecond amino acid mutation is at a position corresponding to residue 42of human IL-2.
 6. The mutant interleukin-2 polypeptide of claim 5,wherein said second amino acid mutation is an amino acid substitution,selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N,F42D, F42R, and F42K.
 7. The mutant interleukin-2 polypeptide of any oneof claims 3 to 6, comprising a third amino acid mutation that abolishesor reduces affinity of the mutant IL-2 polypeptide to the high-affinityIL-2 receptor and preserves affinity of the mutant IL-2 polypeptide tothe intermediate-affinity IL-2 receptor, each compared to a wild-typeIL-2 polypeptide.
 8. The mutant interleukin-2 polypeptide of any one ofclaims 1 to 7, comprising three amino acid mutations that abolish orreduce affinity of the mutant IL-2 polypeptide to the high-affinity IL-2receptor and preserve affinity of the mutant IL-2 polypeptide to theintermediate-affinity IL-2 receptor, each compared to a wild-type IL-2polypeptide, wherein said three amino acid mutations are at positionscorresponding to residue 42, 45, and 72 of human IL-2.
 9. The mutantinterleukin-2 polypeptide of claim 8, wherein said three amino acidmutations are amino acid substitutions selected from the group of F42A,F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S,Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q,L72E, L72N, L72D, L72R, and L72K.
 10. The mutant interleukin-2polypeptide of any one of claims 1 to 9, further comprising an aminoacid mutation which eliminates the O-glycosylation site of IL-2 at aposition corresponding to residue 3 of human IL-2.
 11. The mutantinterleukin-2 polypeptide of any one of claims 1 to 10, wherein saidmutant IL-2 polypeptide is linked to a non-IL-2 moiety.
 12. The mutantinterleukin-2 polypeptide of any one of claims 1 to 11, wherein saidmutant IL-2 polypeptide is linked to a first and a second non-IL-2moiety.
 13. The mutant interleukin-2 polypeptide of claim 12, whereinsaid mutant IL-2 polypeptide shares a carboxy-terminal peptide bond withsaid first non-IL-2 moiety and an amino-terminal peptide bond with saidsecond non-IL-2 moiety.
 14. The mutant interleukin-2 polypeptide of anyone of claims 11 to 13, wherein said non-IL-2 moiety is an antigenbinding moiety.
 15. An immunoconjugate comprising a mutant IL-2polypeptide according to any one of claims 1 to 10 and an antigenbinding moiety.
 16. The immunoconjugate of claim 15, wherein said mutantIL-2 polypeptide shares an amino- or carboxy-terminal peptide bond withsaid antigen binding moiety.
 17. The immunoconjugate of claim 15 or 16,wherein said immunoconjugate comprises as first and a second antigenbinding moiety.
 18. The immunoconjugate of claim 17, wherein said mutantIL-2 polypeptide shares an amino- or carboxy-terminal peptide bond withsaid first antigen binding moiety and said second antigen binding moietyshares an amino- or carboxy-terminal peptide bond with either i) saidmutant IL-2 polypeptide or ii) said first antigen binding moiety. 19.The mutant interleukin-2 polypeptide of claim 14 or the immunoconjugateof any one of claims 15 to 18, wherein said antigen binding moiety is anantibody or an antibody fragment.
 20. The mutant interleukin-2polypeptide of claim 14 or the immunoconjugate of any one of claims 15to 18, wherein said antigen binding moiety is selected from a Fabmolecule and a scFv molecule.
 21. The mutant interleukin-2 polypeptideof claim 14 or the immunoconjugate of any one of claims 15 to 18,wherein said antigen binding moiety is an immunoglobulin molecule,particularly an IgG molecule.
 22. The mutant interleukin-2 polypeptideof claim 14 or the immunoconjugate of any one of claims 15 to 21,wherein said antigen binding moiety is directed to an antigen presentedon a tumor cell or in a tumor cell environment.
 23. The mutantinterleukin-2 polypeptide or immunoconjugate of claim 22, wherein saidantigen is selected from the group of Fibroblast Activation Protein(FAP), the A1 domain of Tenascin-C (TNC A1), the A2 domain of Tenascin-C(TNC A2), the Extra Domain B of Fibronectin (EDB), CarcinoembryonicAntigen (CEA) and the Melanoma-associated Chondroitin SulfateProteoglycan (MCSP).
 24. An isolated polynucleotide encoding the mutantIL-2 polypeptide or immunoconjugate of any one of claims 1 to
 23. 25. Anexpression vector comprising the polynucleotide of claim
 24. 26. A hostcell comprising the polynucleotide of claim 24 or the expression vectorof claim
 25. 27. A method of producing a mutant IL-2 polypeptide or animmunoconjugate thereof, comprising culturing the host cell of claim 26under conditions suitable for the expression of the mutant IL-2polypeptide or the immunoconjugate.
 28. A mutant IL-2 polypeptide orimmunoconjugate produced by the method of claim
 27. 29. A pharmaceuticalcomposition comprising the mutant IL-2 polypeptide or immunoconjugate ofany one of claims 1 to 23 or 28 and a pharmaceutically acceptablecarrier.
 30. The mutant IL-2 polypeptide or immunoconjugate of any oneof claims 1 to 23 or 28 for use in the treatment of a disease in anindividual in need thereof.
 31. The mutant IL-2 polypeptide orimmunoconjugate of claim 30, wherein said disease is cancer.
 32. Use ofthe mutant IL-2 polypeptide or immunoconjugate of any one of claims 1 to23 or 28 for manufacture of a medicament for treating a disease in anindividual in need thereof.
 33. A method of treating disease in anindividual, comprising administering to said individual atherapeutically effective amount of a composition comprising the mutantIL-2 polypeptide or immunoconjugate of any one of claims 1 to 23 or 28in a pharmaceutically acceptable form.
 34. The method of claim 33,wherein said disease is cancer.
 35. A method of stimulating the immunesystem of an individual, comprising administering to said individual aeffective amount of a composition comprising the mutant IL-2 polypeptideor immunoconjugate of any one of claims 1 to 23 or 28 in apharmaceutically acceptable form.
 36. The invention as describedhereinbefore.